Chromosome-specific staining to detect genetic rearrangements associated with chromosome 3 and/or chromosome 17

ABSTRACT

Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

RELATED APPLICATION

This application is a divisional, of application Ser. No. 08/312,914,filed Sep. 30, 1994, now abandoned; which is a continuation ofapplication Ser. No. 08/137,745, filed Oct. 19, 1993, now abandoned,which is a continuation of application Ser. No. 08/015,390, filed Feb.8, 1993, now abandoned, which is a continuation of application Ser. No.07/670,242, filed Mar. 15, 1991, now abandoned, which is acontinuation-in-part of application Ser. No. 07/659,974, filed Feb. 22,1991, now abandoned, which is a continuation-in-part of application Ser.No. 07/537,305, filed Jun. 12, 1990; now abandoned, which is acontinuation-in-part of application Ser. No. 07/497,098, filed Mar. 20,1990, now abandoned, which is a continuation of application Ser. No.07/444,669, filed Dec. 1, 1989, now abandoned, which is a continuationof application Ser. No. 07/382,094, filed Jul. 19, 1989, now abandoned,which is a continuation-in-part of application Ser. No. 06/937,793,filed Dec. 4, 1986, now abandoned, which is a continuation-in-part ofapplication Ser. No. 06/819,314, filed Jan. 16, 1986, now abandoned.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG48 between the U.S. Department of Energy and theUniversity of California, for the operation of Lawrence LivermoreNational Laboratory.

FIELD OF THE INVENTION

The invention relates generally to the field of cytogenetics, and moreparticularly, to the field of molecular cytogenetics. The inventionconcerns methods for identifying and classifying chromosomes. Still moreparticularly, this invention concerns nucleic acid probes which can bedesigned by the processes described herein to produce stainingdistributions that can extend along one or more whole chromosomes,and/or along a region or regions on one or more chromosomes, includingstaining patterns that extend over the whole genome. Staining patternscan be tailored for any desired cytogenetic application, includingprenatal, tumor and disease related cytogenetic applications, amongothers. The invention provides for compositions of nucleic acid probesand for methods of staining chromosomes therewith to identify normalchromosomes and chromosomal abnormalities in metaphase spreads and ininterphase nuclei. The probe-produced staining patterns of thisinvention facilitate the microscopic and/or flow cytometricidentification of normal and abnormal chromosomes and thecharacterization of the genetic nature of particular abnormalities. Theparticular focus of this application is that wherein the abnormalitiesare genetic rearrangements.

Although most of the examples herein concern human chromosomes and muchof the language herein is directed to human concerns, the concept ofusing nucleic acid probes for staining or painting chromosomes isapplicable to chromosomes from any source including both plants andanimals.

BACKGROUND OF THE INVENTION

Chromosome abnormalities are associated with genetic disorders,degenerative diseases, and exposure to agents known to causedegenerative diseases, particularly cancer, German, “Studying HumanChromosomes Today,” American Scientist Vol. 58, pgs. 182-201 (1970);Yunis, “The Chromosomal Basis of Human Neoplasia,” Science Vol. 221,pgs. 227-236 (1983); and German, “Clinical Implication of ChromosomeBreakage,” in Genetic Damage in Man Caused by Environmental Agents,Berg, Ed., pgs. 65-86 (Academic Press, New York, 1979). Chromosomalabnormalities can be of several types, including: extra or missingindividual chromosomes, extra or missing portions of a chromosome(segmental duplications or deletions), breaks, rings and chromosomalrearrangements, among others. Chromosomal or genetic rearrangementsinclude translocations (transfer of a piece from one chromosome ontoanother chromosome), dicentrics (chromosomes with two centromeres),inversions (reversal in polarity of a chromosomal segment), insertions,amplifications, and deletions.

Detectable chromosomal abnormalities occur with a frequency of one inevery 250 human births. Abnormalities that involve deletions oradditions of chromosomal material alter the gene balance of an organismand generally lead to fetal death or to serious mental and physicaldefects. Down syndrome can be caused by having three copies ofchromosome 21 instead of the normal 2. This syndrome is an example of acondition caused by abnormal chromosome number, or aneuploidy. Downsyndrome can also be caused by a segmental duplication of a subregion onchromosome 21 (such as, 21q22), which can be present on chromosome 21 oron another chromosome. Edward syndrome (18+), Patau syndrome (13+),Turner syndrome (XO) and Kleinfelter syndrome (XXY) are among the mostcommon numerical aberrations. [Epstein, The Consequences of ChromosomeImbalance: Principles, Mechanisms and Models (Cambridge Univ. Press1986); Jacobs, Am. J. Epidemiol, 105:180 (1977); and Lubs et al.,Science, 169:495 (1970).]

Retinoblastoma (del 13q14), Prader-Willi syndrome (del 15q11-q13),Wilm's tumor (del 11p13) and Cri-du-chat syndrome (del 5p) are examplesof important disease linked structural aberrations. [Nora and Fraser,Medical Genetics: Principles and Practice. (Lea and Febiger 1989).]

Measures of the frequency of structurally aberrant chromosomes, forexample, dicentric chromosomes, caused by clastogenic agents, such as,ionizing radiation or chemical mutagens, are widely used as quantitativeindicators of genetic damage caused by such agents, BiochemicalIndicators of Radiation Injury in Man (International Atomic EnergyAgency, Vienna, 1971); and Berg, Ed. Genetic Damage in Man Caused byEnvironmental Agents (Academic Press, New York, 1979). A host ofpotentially carcinogenic and teratogenic chemicals are widelydistributed in the environment because of industrial and agriculturalactivity. These chemicals include pesticides, and a range of industrialwastes and by-products, such as halogenated hydrocarbons, vinylchloride, benzene, arsenic, and the like, Kraybill et al., Eds.,Environmental Cancer (Hemisphere Publishing Corporation, New York,1977). Sensitive measures of chromosomal breaks and other abnormalitiescould form the basis of improved dosimetric and risk assessmentmethodologies for evaluating the consequences of exposure to suchoccupational and environmental agents.

Current procedures for genetic screening and biological dosimetryinvolve the analysis of karyotypes. A karyotype is the particularchromosome complement of an individual or of a related group ofindividuals, as defined both by the number and morphology of thechromosomes usually in mitotic metaphase. It includes such things astotal chromosome number, copy number of individual chromosome types(e.g., the number of copies of chromosome X), and chromosomalmorphology, e.g., as measured by length, centromeric index,connectedness, or the like. Chromosomal abnormalities can be detected byexamination of karyotypes. Karyotypes are conventionally determined bystaining an organism's metaphase, or otherwise condensed (for example,by premature chromosome condensation) chromosomes. Condensed chromosomesare used because, until recently, it has not been possible to visualizeinterphase chromosomes due to their dispersed condition and the lack ofvisible boundaries between them in the cell nucleus.

A number of cytological techniques based upon chemical stains have beendeveloped which produce longitudinal patterns on condensed chromosomes,generally referred to as bands. The banding pattern of each chromosomewithin an organism usually permits unambiguous identification of eachchromosome type, Latt, “Optical Studies of Metaphase ChromosomeOrganization,” Annual Review of Biophysics and Bioengineering, Vol. 5,pgs. 1-37 (1976). Accurate detection of some important chromosomalabnormalities, such as translocations and inversions, has required suchbanding analysis.

Unfortunately, such conventional banding analysis requires cellculturing and preparation of high quality metaphase spreads, which istime consuming and labor intensive, and frequently difficult orimpossible. For example, cells from many tumor types are difficult toculture, and it is not clear that the cultured cells are representativeof the original tumor cell population. Fetal cells capable of beingcultured need to be obtained by invasive means and need to be culturedfor several weeks to obtain enough metaphase cells for analysis. In manycases, the banding patterns on the abnormal chromosomes do not permitunambiguous identification of the portions of the normal chromosomesthat make them up. Such identification may be important to indicate thelocation of important genes involved in the abnormality. Further, thesensitivity and resolving power of current methods of karyotyping arelimited by the fact that multiple chromosomes or chromosomal regionshave highly similar staining characteristics, and that abnormalities(such as deletions) which involve only a fraction of a band are notdetectable. Therefore, such methods are substantially limited for thediagnosis and detailed analysis of contiguous gene syndromes, such aspartial trisomy, Prader-Willi syndrome [Emanuel, Am. J. Hum. Genet.43:575 (1988); Schmickel, J. Pediatr., 109:231 (1986)] andretinoblastoma [Sparkes, Biochem. Biophys. Acta., 780:95 (1985)].

Thus, conventional banding analysis has several important limitations,which include the following. 1) It is labor intensive, time consuming,and requires a highly trained analyst. 2) It can be applied only tocondensed chromosomes. 3) It does not allow for the detection ofstructural aberrations involving less than 3-15 megabases (Mb),depending upon the nature of the aberration and the resolution of thebanding technique [Landegren et al., Science 242:229 (1988)]. Thisinvention provides for probe compositions and methods to overcome suchlimitations of conventional banding analysis.

The chemical staining procedures of the prior art provide patterns overa genome for reasons not well understood and which cannot be modified asrequired for use in different applications. Such chemical stainingpatterns were used to map the binding site of probes. However, onlyoccasionally, and with great effort, was in situ hybridization used toobtain some information about the position of a lesion, for example, abreakpoint relative to a particular DNA sequence. The present inventionovercomes the inflexibility of chemical staining in that it stains agenome in a pattern based upon nucleic acid sequence; therefore thepattern can be altered as required by changing the nucleic acid sequenceof the probe. The probe-produced staining patterns of this inventionprovide reliable fundamental landmarks which are useful in cytogeneticanalysis.

Automated detection of structural abnormalities of chromosomes withimage analysis of chemically stained bands would require the developmentof a system that can detect and interpret the banding patterns producedon metaphase chromosomes by conventional techniques. It has proven to bevery difficult to identify reliably by automated means normalchromosomes that have been chemically stained; it is much more difficultto differentiate abnormal chromosomes having structural abnormalities,such as, translocations. Effective automated detection of translocationsin conventionally banded chromosomes has not been accomplished afterover a decade of intensive work. The probe-produced banding patterns ofthis invention are suitable for such automated detection and analysis.

In recent years rapid advances have taken place in the study ofchromosome structure and its relation to genetic content and DNAcomposition. In part, the progress has come in the form of improvedmethods of gene mapping based on the availability of large quantities ofpure DNA and RNA fragments for probes produced by genetic engineeringtechniques, e.g., Kao, “Somatic Cell Genetics and Gene Mapping,”International Review of Cytology Vol. 85, pgs. 109-146 (1983), andD'Eustachio et al., “Somatic Cell Genetics in Gene Families,” Science,Vol. 220, pgs. 9, 19-924 (1983). The probes for gene mapping compriselabeled fragments of single-stranded or double-stranded DNA or RNA whichare hybridized to complementary sites on chromosomal DNA. With suchprobes it has been crucially important to produce pure, or homogeneous,probes to minimize hybridizations at locations other than at the site ofinterest, Henderson, “Cytological Hybridization to MammalianChromosomes,” International Review of Cytology Vol. 76, pgs. 1-46(1982).

The hybridization process involves unravelling, or melting, thedouble-stranded nudeic acids of the probe and target by heating, orother means (unless the probe and target are single-stranded nucleicacids). This step is sometimes referred to as denaturing the nudeicacid. When the mixture of probe and target nucleic acids cool, strandshaving complementary bases recombine, or anneal. When a probe annealswith a target nudeic acid, the probe's location on the target can bedetected by a label carried by the probe or by some intrinsiccharacteristics of the probe or probe-target duplex. When the targetnucleic acid remains in its natural biological setting, e.g., DNA inchromosomes, mRNA in cytoplasm, portions of chromosomes or cell nuclei(albeit fixed or altered by preparative techniques), the hybridizationprocess is referred to as in situ hybridization.

In situ hybridization probes were initially limited to identifying thelocation of genes or other well defined nucleic acid sequences onchromosomes or in cells. Comparisons of the mapping of single-copyprobes to normal and abnormal chromosomes were used to examinechromosomal abnormalities. Cannizzaro et al., Cytogenetics and CellGenetics, 39:173-178 (1985). Distribution of the multiple binding sitesof repetitive probes could also be determined.

Hybridization with probes which have one target site in a haploidgenome, single-copy or unique sequence probes, has been used to map thelocations of particular genes in the genome [Harper and Saunders,“Localization of the Human Insulin Gene to the Distal End of the ShortArm of Chromosome 11,” Proc. Natl. Acad. Sci., Vol. 78, pgs. 4458-4460(1981); Kao et al., “Assignment of the Structural Gene Coding forAlbumin to Chromosome 4,” Human Genetics, Vol. 62, pgs. 337-341 (1982)];but such hybridizations are not reliable when the size of the targetsite is small. As the amount of target sequence for low complexitysingle-copy probes is small, only a portion of the potential targetsites in a population of cells form hybrids with the probe. Therefore,mapping the location of the specific binding site of the probe has beencomplicated by background signals produced by non-specific binding ofthe probe and also by noise in the detection system (for example,autoradiography or immunochemistry). The unreliability of signals forsuch prior art single-copy probes has required statistical analysis ofthe positions of apparent hybridization signals in multiple cells to mapthe specific binding site of the probe.

Wallace et al., in “The Use of Synthetic Oligonucleotides asHybridization Probes. II. Hybridization of Oligonudeotides of MixedSequence to Rabbit Beta-Globin DNA,” Nucleic Acids Research, Vol. 9,pgs. 879-894 (1981), disclose the construction of syntheticoligonucleotide probes having mixed base sequences for detecting asingle locus corresponding to a structural gene. The mixture of basesequences was determined by considering all possible nucleotidesequences which could code for a selected sequence of amino acids in theprotein to which the structural gene corresponded.

Olsen et al., in “Isolation of Unique Sequence Human X ChromosomalDeoxyribonucleic Acid,” Biochemistry, Vol. 19, pgs. 2419-2428 (1980),disclose a method for isolating labeled unique sequence human Xchromosomal DNA by successive hybridizations: first, total genomic humanDNA against itself so that a unique sequence DNA fraction can beisolated; second, the isolated unique sequence human DNA fractionagainst mouse DNA so that homologous mouse/human sequences are removed;and finally, the unique sequence human DNA not homologous to mouseagainst the total genomic DNA of a human/mouse hybrid whose only humanchromosome is chromosome X, so that a fraction of unique sequence Xchromosomal DNA is isolated. Individual clones are then isolated fromthis fraction and are candidates for human X chromosome specific DNAsequences.

Manuelidis et al., in “Chromosomal and Nuclear Distribution of the HindIII 1.9-KB Human DNA Repeat Segment,” Chromosoma Vol. 91, pp. 28-38(1984), disclose the construction of a single kind of DNA probe fordetecting multiple loci on chromosomes corresponding to the location ofmembers of a family of repeated DNA sequences. Such probes are hereintermed repetitive probes.

Different repetitive sequences may have different distributions onchromosomes. They may be spread over all chromosomes as in the justcited reference, or they may be concentrated in compact regions of thegenome, such as, on the centromeres of the chromosomes, or they may haveother distributions. In some cases, such a repetitive sequence ispredominantly located on a single chromosome, and therefore is achromosome-specific repetitive sequence. [Willard et al., “Isolation andCharacterization of a Major Tandem Repeat Family from the Human XChromosome,” Nucleic Acids Research, Vol. 11, pgs. 2017-2033 (1983).]

A probe for repetitive sequences shared by all chromosomes can be usedto discriminate between chromosomes of different species if the sequenceis specific to one of the species. Total genomic DNA from one specieswhich is rich in such repetitive sequences can be used in this manner.[Pinkel et al. (III), PNAS USA, 83:2934 (1986); Manuelidis, Hum. Genet.,71:288 (1985) and Durnam et al., Somatic Cell Molec. Genet., 11:571(1985.]

Recently, there has been an increased availability of probes forrepeated sequences (repetitive probes) that hybridize intensely andspecifically to selected chromosomes. [Trask et al., Hum. Genet., 78:251(1988) and references cited therein.] Such probes are now available forover half of the human chromosomes. In general, they bind to repeatedsequences on compact regions of the target chromosome near thecentromere. However, one probe has been reported that hybridizes tohuman chromosome 1p36, and there are several probes that hybridize tohuman chromosome Yq. Hybridization with such probes permits rapididentification of chromosomes in metaphase spreads, determination of thenumber of copies of selected chromosomes in interphase nuclei [Pinkel etal. (I), PNAS USA, 83:2934 (1986); Pinkel et al. (II), Cold SpringHarbor Symp. Quant. Biol., 51:151 (1986) and Cremer et al., Hum. Genet,74:346 (1986)] and determination of the relative positions ofchromosomes in interphase nuclei [Trask et al., supra; Pinkel et al.(I), supra; Pinkel et al. (II), supra; Manuelidis, PNAS USA, 81:3123(1984); Rappold et al., Hum. Genet., 67:317 (1984); Schardin et al.,Hum. Genet., 71:282 (1985); and Manuelidis, Hum. Genet., 71:288 (1985)].

However, many applications are still limited by the lack of appropriateprobes. For example, until the methods described herein were invented,probes with sufficient specificity for prenatal diagnosis were notavailable for chromosome 13 or 21. Further, repetitive probes are notvery useful for detection of structural aberrations since theprobability is low that the aberrations will involve the region to whichthe probe hybridizes.

This invention overcomes the prior art limitations on the use of probesand dramatically enhances the application of in situ hybridization forcytogenetic analysis. As indicated above, prior art probes have not beenuseful for in-depth cytogenetic analysis. Low complexity single-copyprobes do not at this stage of hybridization technology generatereliable signals. Although repetitive probes do provide reliablesignals, such signals cannot be tailored for different applicationsbecause of the fixed distribution of repetitive sequences in a genome.The probes of this invention combine the hybridization reliability ofrepetitive probes with the flexibility of being able to tailor thebinding pattern of the probe to any desired application.

The enhanced capabilities of the probes of this invention come fromtheir increased complexity. Increasing the complexity of a probeincreases the probability, and therefore the intensity, of hybridizationto the target region, but also increases the probability of non-specifichybridizations resulting in background signals. However, within theconcept of this invention, it was considered that such backgroundsignals would be distributed approximately randomly over the genome.Therefore, the net result is that the target region could be visualizedwith increased contrast against such background signals.

Exemplified herein are probes in an approximate complexity range of fromabout 50,000 bases (50 kb) to hundreds of millions of bases. Suchrepresentative probes are for compact loci and whole human chromosomes.Prior to this invention, probes employed for in situ hybridizationtechniques had complexities below 40 kb, and more typically on the orderof a few kb.

Staining chromosomal material with the probes of this invention issignificantly different from the chemical staining of the prior art. Thespecificity of the probe produced staining of this invention arises froman entirely new source—the nucleic acid sequences in a genome. Thus,staining patterns of this invention can be designed to highlightfundamental genetic information important to particular applications.

The procedures of this invention to construct probes of any desiredspecificity provide significant advances in a broad spectrum ofcytogenetic studies. The analysis can be carried out on metaphasechromosomes and interphase nuclei. The techniques of this invention canbe especially advantageous for applications where high-quality bandingby conventional methods is difficult or suspected of yielding biasedinformation, e.g., in tumor cytogenetics. Reagents targeted to sites oflesions known to be diagnostically or prognostically important, such astumor type-specific translocations and deletions, among other tumorspecific genetic arrangements, permit rapid recognition of suchabnormalities. Where speed of analysis is the predominant concern, e.g.,detection of low-frequency chromosomal aberrations induced by toxicenvironmental agents, the compositions of this invention permit adramatic increase in detection efficiency in comparison to previoustechniques based on conventional chromosome banding.

Further, prenatal screening for disease-linked chromosome aberrations(e.g., trisomy 21) is enhanced by the rapid detection of suchaberrations by the methods and compositions of this invention.Interphase aneuploidy analysis according to this invention isparticularly significant for prenatal diagnosis in that it yields morerapid results than are available by cell culture methods. Further, fetalcells separated from maternal blood, which cannot be cultured by routineprocedures and therefore cannot be analysed by conventional karyotypingtechniques, can be examined by the methods and compositions of thisinvention. In addition, the intensity, contrast and color combinationsof the staining patterns, coupled with the ability to tailor thepatterns for particular applications, enhance the opportunities forautomated cytogenetic analysis, for example, by flow cytometry orcomputerized microscopy and image analysis.

This application specifically claims chromosome specific reagents forthe detection of genetic rearrangements and methods of using suchreagents to detect such rearrangements. Representative geneticrearrangements so detected are those that produce a fusiongene—BCR-ABL—that is diagnostic for chronic myelogenous leukemia (CML)and those associated with chromosomes 3, 13 (retinoblastoma genetherein), and 17, such as, deletions, amplifications and translocationsthereof.

Chronic myelogenous leukemia (CML) is a neoplastic proliferation of bonemarrow cells genetically characterized by the fusion of the BCR and ABLgenes on chromosomes 9 and 22. That fusion usually involves a reciprocaltranslocation t(9;22)(q34;q11), which produces the cytogeneticallydistinctive Philadelphia chromosome (Ph¹). However, more complexrearrangements may cause BCR-ABL fusion. At the molecular level, fusioncan be detected by Southern analysis or by in vitro amplification of themRNA from the fusion gene using the polymerase chain reaction (PCR).Those techniques are sensitive but cannot be applied to single cells.

Clearly, a sensitive method for detecting chromosomal abnormalities and,more specifically, genetic rearrangements, such as, for example, thetumor specific arrangements associated with CML, the chromosome 3 and 17deletions, amplifications and translocations associated with variouscancers, and those associated with the retinoblastoma gene, would be ahighly useful tool for genetic screening. This invention provides suchtools.

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Fusion of the proto-oncogene c-ABL from the long arm of chromosome 9with the BCR gene of chromosome 22 is a consistent finding in CML (1-3).That genetic change leads to formation of a BCR-ABL transcript that istranslated to form a 210 kd protein present in virtually all cases ofCML (46). In 90% of the cases, the fusion gene results from a reciprocaltranslocation involving chromosomes 9 and 22 producing a cytogeneticallydistinct small acrocentric chromosome called the Philadelphia (Ph¹)chromosome (7-12), FIG. 8. However, standard cytogenetics does not havethe resolution to distinguish closely spaced breakpoints, such as thosecharacteristic of CML and acute lymphocytic leukemia (ALL), and missesfusions produced by more complex rearrangements. Mapping and cloning ofthe breakpoint regions in both genes has lead to molecular techniquescapable of demonstrating BCR-ABL fusion in CML cases where the Ph¹chromosome could not be detected cytogenetically (13-16). Southernanalysis for BCR rearrangements has become the standard for diagnosis ofCML. More recently, fusion has been detected by in vitro amplificationof a cDNA transcript copied from CML mRNA using reverse transcriptase(17-23). That technique permits detection of BCR-ABL transcript from CMLcells present at low frequencies. Both of those techniques utilizenucleic acid obtained from cell populations so that correlation betweengenotype and phenotype for individual cells is not possible.

Chromosomal deletions involving tumor suppressor genes may play animportant role in the development and progression of solid tumors. Theretinoblastoma gene (Rb-1), located in chromosome 13q14, is the mostextensively characterized tumor suppressor gene (Friend et al., 1986;Lee et al., 1987; Fung et al., 1987). The Rb-1 gene product, a 105 kDanuclear phosphoprotein, apparently plays an important role in cell cycleregulation (Lee et al., 1987; Howe et al., 1990). Altered or lostexpression of the Rb protein is caused by inactivation of both genealleles either through a point mutation or a chromosomal deletion. Rb-1gene alterations have been found not only in retinoblastomas (Friend etal., 1986; Lee et al., 1987; Fung et al., 1987) but also in othermalignancies such as osteosarcomas (Friend et al. 1986), small cell lungcancer (Hensel et al., 1990; Rygaard et al., 1990) and breast cancer(Lee et al., 1988; T'Ang et al., 1988; Varley et al., 1989). Restrictionfragment length polymorphism (RFLP) studies have indicated that suchtumor types have frequently lost heterozygosity at 13q suggesting thatone of the Rb-1 gene alleles has been lost due to a gross chromosomaldeletion (Lundberg et al., 1987; Bowcock et al., 1990).

Section IX infra describes the use of fourteen lambda phage clonesspanning all the exons of the Rb-1 gene region, about 150 kb of genomicDNA, as a high complexity probe for chromosome-specific painting. Anintense signal produced in metaphase chromosomes confirmed the locationof the Rb-1 gene at chromosome 13q14. Two Rb-1 hybridization signalswere detected in about 90% of normal interphase nuclei, whereas two celllines having a cytogenetically defined deletion involving the Rb-1 generegion showed only one hybridization signal. Gene deletion was confirmedby analyzing metaphase spreads from these cell lines cohybridized withchromosome 13/21 alpha satellite probe. Also analyzed were touchpreparations and fine needle aspirates of breast carcinomas;heterogeneity was shown in Rb-1 gene copy number both within and betweentumors.

Genetic rearrangements involving only subregions of the Rb-1 gene havebeen described (Bookstein et al., 1988; Canning and Dryja, 1989). Theinventors hereof, to detect such subregions of the Rb-1 gene, usedsmaller probes comprising 1-5 contiguous lambda phage clones to stainspecific subregions within the Rb-1 gene thus allowing detection ofaberrations within that tumor suppressor gene. Such representativeexamples of the chromosome-specific staining methods of this inventionprovide information on actual gene copy numbers and rearrangements fromindividual morphologically defined tumor cells useful in the evaluationof neoplasia-associated gene aberrations as well as intratumor geneticheterogeneity.

The deletion of the short arm of chromosome 3 has been associated withseveral cancers, for example, small cell lung cancer, renal and ovariancancers; it has been postulated that one or more putative tumorsuppressor genes is or are located in the p region of chromosome 3 (ch.3p) [Minna et al., Symposia on Quantitative Biology, Vol. L1:843-853(CSH Lab 1986); Cohen et al., N. Eng. J. Med., 301:592-595 (1979);Bergerham et al., Cancer Res., 49:1390-1396 (1989); Whang-Peng et al.,Can. Genet. Cytogenet, 11:91-106 (1984); and Trent et al., Can. Genet.Cytogenet, 14:153-161 (1985)]. As shown in Section X infrachromosome-specific staining according to this invention can be used tocreate bands of stained nucleic acid that detect structural aberrations,for example, those of chromosome 3. The examples of Section Xdemonstrate the initial stages of a probe-based banding pattern todetect genetic rearrangements of chromosome 3.

Described herein are chromosome-specific reagents and methods to detectgenetic rearrangements, such as those exemplified herein for the BCR-ABLfusion, deletions, amplifications, and translocations of chromosomes 3,17 and 13, that supply information unavailable by existing techniques.

SUMMARY OF THE INVENTION

This invention concerns methods of staining chromosomal material basedupon nucleic acid sequence that employ one or more nucleic acid probes.Said methods produce staining patterns that can be tailored for specificcytogenetic analyses. It is further an object of this invention toproduce nucleic acid probes that are useful for cytogenetic analysis,that stain chromosomal material with reliable signals. Such probes areappropriate for in situ hybridization. Preferred nucleic acid probes forcertain applications of this invention are those of sufficientcomplexity to stain reliably each of two or more target sites.

The invention provides methods and compositions for staining chromosomalmaterial. The probe compositions of this invention at the current stateof hybridization techniques are typically of high complexity, usuallygreater than about 50 kb of complexity, the complexity depending uponthe application for which the probe is designed. In particular,chromosome specific staining reagents are provided which compriseheterogeneous mixtures of nucleic acid fragments, each fragment having asubstantial fraction of its sequences substantially complementary to aportion of the nucleic acid for which specific staining is desired—thetarget nucleic acid, preferably the target chromosomal material. Ingeneral, the nucleic acid fragments are labeled by means as exemplifiedherein and indicated infra. However, the nucleic acid fragments need notbe directly labeled in order for the binding of probe fragments to thetarget to be detected; for example, such nudeic acid binding can bedetected by anti-RNA/DNA duplex antibodies and antibodies to thymidinedimers. The nucleic acid fragments of the heterogenous mixtures includedouble-stranded or single-stranded RNA or DNA.

This invention concerns chromosome specific reagents and methods ofstaining targeted chromosomal material that is in the vicinity of asuspected genetic earrangement. Such genetic rearrangement include butare not limited to translocations, inversions, insertions,amplifications and deletions. Aneuploidy is included herein in the term“amplifications”. When such a genetic rearrangement is associated with adisease, such chromosome specific reagents are referred to as diseasespecific reagents or probes. When such a genetic rearrangement isassociated with cancer, such reagents are referred to as tumor specificreagents or probes.

This invention provides for nucleic acid probes that reliably staintargeted chromosomal materials in the vicinity of one or more suspectedgenetic rearrangements. Such nucleic acid probes useful for thedetection of genetic rearrangements are typically of high complexity.Such nucleic acid probes preferably comprise nucleic acid sequences thatare substantially homologous to nudeic acid sequences in chromosomalregions that flank and/or extend partially or fully across breakpointsassociated with genetic rearrangements.

This invention further provides for methods and reagents to distinguishbetween cytogenetically similar but genetically different chromosomalrearrangements.

Specifically herein exemplified are chromosome specific regents andmethods to detect genetic rearrangements, e.g., translocations,amplifications and insertions, that produce the BCR-ABL fusion which isdiagnostic for chronic myelogenous leukemia (CML). Such chromosomespecific reagents for the diagnosis of CML contain nucleic acidsequences which are substantially homologous to chromosomal sequences inthe vicinity of the translocation breakpoint regions of chromosomalregions 9q34 and 22q11 associated with CML.

Those reagents produce a staining pattern which is distinctively alteredwhen the BCR-ABL fusion characteristic of CML occurs. FIG. 11graphically demonstrates a variety of staining patterns which, alongwith other potential staining patterns, are altered in the presence of agenetic rearrangement, such as, the BCR-ABL fusion.

The presence of a genetic rearrangement can be determined by applyingthe reagents of this invention according to methods herein described andobserving the proximity of and/or other characteristics of the signalsof the staining patterns produced.

Preferably, the chromosome specific reagents used to detect CML of thisinvention comprise nudeic acid sequences having a complexity of fromabout 50 kilobases (kb) to about 1 megabase (Mb), more preferably fromabout 50 kb to about 750 kb, and still more preferably from about 200 kbto about 400 kb.

This invention further provides for methods of distinguishing betweensuspected genetic rearrangements that occur in relatively closeproximity in a genome wherein the chromosome specific reagents comprisenucleic acid sequences substantially homologous to nudeic acid sequencesin the vicinity of said suspected genetic rearrangements. An example ofsuch a differentiation between two potential genetic rearrangements isthe differential diagnosis of CML from acute lymphocytic leukemeia(ALL).

This invention still further provides methods and reagents for producingstaining patterns in a patient who is afflicted with a diseaseassociated genetic rearrangement, such as those associated with theBCR-ABL fusion in CML, wherein said staining patterns are predictiveand/or indicative of the response of a patient to various therapeuticregimens, such as chemotherapy, radiation, surgery, and transplantation,such as bone marrow transplantation. Such staining patterns can beuseful in monitoring the status of such a patient, preferably on a cellby cell basis, and can be predictive of a disease recurrence for apatient that is in remission. Computer assisted microscopic analysis canassist in the interpretation of staining patterns of this invention, andthe invention provides for methods wherein computer assisted microscopicanalysis is used in testing patient cells on a call by cell basis, fore.g., to search for residual disease in a patient.

Still further, this invention provides for methods and reagents todetermine the molecular basis of genetic disease, and to detect specificgenetically based diseases.

Still further, this invention provides for methods and reagents fordetecting contiguous gene syndromes comprising the in situ hybridizationof nucleic acid probes which comprise sequences which are substantiallyhomologous to nudeic acid sequences characteristic of one or morecomponents of a contiguous gene syndrome. Representative of such acontiguous gene syndrome is Down syndrome.

Also provided are methods of simultaneously detecting geneticrearrangements of multiple loci in a genome comprising in situhybridization of high complexity nucleic acid probes comprising nucleicacid sequences that are substantially homologous to nucleic acidsequences in multiple loci in a genome.

Still further provided are methods of searching for geneticrearrangements in a genome. For example, conventional banding analysismay indicate an abnormality in a chromosomal region of a genome underexamination. Methods of this invention may include the application ofnucleic acid probes, produced from the vicinity of that chromosomalregion of a normal genome, by in situ hybridization to cells containingthe abnormality to detail the exact location and kind of geneticrearrangement of said abnormality by observation of the stainingpatterns so produced.

The invention still further provides for high complexity nucleic acidprobes which have been optimized for rapid, efficient and automateddetection of genetic rearrangements.

One way to produce a probe of high complexity is to pool several or manyclones, for example, phage, plasmid, cosmid, and/or YAC clones, amongothers, wherein each clone contains an insert that is capable ofhybridizing to some part of the target in a genome. Another way toproduce such a probe is to use the polymerase chain reaction (PCR).

Heterogeneous in reference to the mixture of labeled nucleic acidfragments means that the staining reagents comprise many copies each offragments having different sequences and/or sizes (e.g., from thedifferent DNA clones pooled to make the probe). In preparation for use,these fragments may be cut, randomly or specifically, to adjust the sizedistribution of the pieces of nucleic acid participating in thehybridization reaction.

As discussed more fully below, preferably the heterogeneous probemixtures are substantially free from nucleic acid sequences withhybridization capacity to non-target nucleic acid. Most of suchsequences bind to repetitive sequences which are shared by the targetand non-target nucleic acids, that is, shared repetitive sequences.

Methods to remove undesirable nucleic acid sequences and/or to disablethe hybridization capacity of such sequences are discussed more fullybelow. [See Section II]. Such methods include but are not limited to theselective removal or screening of shared repetitive sequences from theprobe; careful selection of nucleic acid sequences for inclusion in theprobe; blocking shared repetitive sequences by the addition of unlabeledgenomic DNA, or, more carefully selecting nucleic acid sequences forinclusion in the blocking mixture; incubating the probe mixture forsufficient time for reassociation of high copy repetitive sequences, orthe like.

Preferably, the staining reagents of the invention are applied tointerphase or metaphase chromosomal DNA by in situ hybridization, andthe chromosomes are identified or classified, i.e., karyotyped, bydetecting the presence of the label, such as biotin or ³H, on thenucleic acid fragments comprising the staining reagent.

The invention includes chromosome staining reagents for the totalgenomic complement of chromosomes, staining reagents specific to singlechromosomes, staining reagents specific to subsets of chromosomes, andstaining reagents specific to subregions within single or multiplechromosomes. The term “chromosome-specific,” is understood to encompassall of these embodiments of the staining reagents of the invention. Theterm is also understood to encompass staining reagents made from anddirected against both normal and abnormal chromosome types.

A preferred method of making the chromosome-specific staining reagentsof the invention includes: 1) isolating chromosomal DNA from aparticular chromosome type or target region or regions in the genome, 2)amplifying the isolated DNA to form a heterogeneous mixture of nucleicacid fragments, 3) disabling the hybridization capacity of or removingshared repeated sequences in the nucleic acid fragments, and 4) labelingthe nucleic acid fragments to form a heterogeneous mixture of labelednucleic acid fragments. As described more fully below, the ordering ofthe steps for particular embodiments varies according to the particularmeans adopted for carrying out the steps.

The present invention addresses problems associated with karyotypingchromosomes, especially for diagnostic and dosimetric applications. Inparticular, the invention overcomes problems which arise because of thelack of stains that are sufficiently chromosome-specific by providingreagents comprising heterogeneous mixtures of nudeic acid fragments thatcan be hybridized to the target DNA and/or RNA, e.g., the targetchromosomes, target subsets of chromosomes, or target regions ofspecific chromosomes. The staining technique of the invention opens upthe possibility of rapid and highly sensitive detection of chromosomalabnormalities, particularly genetic rearrangements, in both metaphaseand interphase cells using standard clinical and laboratory equipmentand improved analysis using automated techniques. It has directapplication in genetic screening, cancer diagnosis, and biologicaldosimetry.

This invention further specifically provides for methods and nucleicacid probes for staining fetal chromosomal material, whether condensed,as in metaphase, or dispersed as in interphase. Still further, theinvention provides for a non-embryo-invasive method of karyotyping thechromosomal material of fetal cells, wherein the fetal cells have beenseparated from maternal blood. Such fetal cells are preferablyleukocytes and/or cytotrophoblasts. Exemplary nudeic acid probes arehigh complexity probes chromosome-specific for chromosome types 13, 18and/or 21. Representative probes comprise chromosome-specific Bluescribeplasmid libraries from which a sufficient number of shared repetitivesequences have been removed or the hybridization capacity thereof hasbeen disabled prior to and/or during hybridization with the target fetalchromosomes.

This invention still further provides for test kits comprisingappropriate nucleic acid probes for use in tumor cytogenetics, in thedetection of disease related loci, in the analysis of structuralabnormalities, for example translocations, among other geneticrearrangements, and for biological dosimetry.

This invention further provides for prenatal screening kits comprisingappropriate nudeic acid probes of this invention. This invention alsoprovides for test kits comprising high complexity probes for thedetection of genetic rearrangements, and specifically for thoseproducing the BCR-ABL fusion characteristic of CML.

The methods and compositions of this invention permit staining ofchromosomal material with patterns appropriate for a desiredapplication. The pattern may extend over some regions of one or morechromosomes, or over some or all the chromosomes of a genome and maycomprise multiple distinguishable sections, distinguishable, forexample, by multiple colors. Alternatively, the pattern may be focusedon a particular portion or portions of a genome, such as a portion orportions potentially containing a deletion or breakpoint that isdiagnostically or prognostically important for one or more tumors, or onthose portions of chromosomes having significance for prenataldiagnosis.

The staining patterns may be adjusted for the analysis method employed,for example, either a human observer or automated equipment, such as,flow cytometers or computer assisted microscopy. The patterns may bechosen to be appropriate for analysis of condensed chromosomes ordispersed chromosomal material.

It is further an object of the instant invention to detect smallspecific deletions invisible by conventional banding analyses, includingsubregion deletions within a gene, for example, the Rb-1 gene.

It is still further an object of this invention to provide methods ofstudying the genetic heterogeneity of cancers, such as, solid tumors,and of gene copy numbers and structural abnormalities frommorphologically defined individual tumor cells.

The invention further provides for automated means of detecting andanalyzing chromosomal abnormalities, particularly geneticrearrangements, as indicated by the staining patterns produced accordingto this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, B and C and FIGS. 2A and 2B illustrate the hybridization of achromosome-specific 21 library to human metaphase spread wherein theinserts were cloned in Lambda phage Charon 21A. The hybridizationcapacity of the high copy repetitive sequences in the library wasreduced by the addition of unlabeled genomic DNA to the hybridizationmixture. The probe was labeled with biotin, which was detected withgreen FITC-avidin (fluorescein isothiocyanate avidin). All of the DNA inthe chromosomes was stained with the blue fluorescent dye DAPI(4,6-diamidino-2-phenylindole).

FIG. 1A is a binary image of the DAPI stain in the human metaphasespread obtained by using a TV camera attached to a fluorescencemicroscope. Filters appropriate for DAPI visualization were used.Computer processing of the image shows all portions above a chosenthreshold intensity as white, and the rest as black.

FIG. 1B is a binary image of the FITC staining of the same humanmetaphase spread as in FIG. 1A. The image was processed as in FIG. 1Abut the filter was changed in the microscope such that the FITC attachedto the probe is visible rather than the DAPI.

FIG. 1C is a binary image of the chromosome 21s alone, nonspecificallystained objects (which are smaller) having been removed by standardimage processing techniques on the binary image of FIG. 1B.

FIG. 2A is a color photograph of the DAPI stain in a human metaphasespread which was prepared and hybridized contemporaneously with thespread shown in the computer generated binary images of FIGS. 1A, B andC.

FIG. 2B is a color photograph of the fluorescein attached to the DNAprobe in the same human metaphase spread as shown in FIG. 2A. It wasobtained by changing the filters in the fluorescence microscope toexcite fluorescein rather than DAPI. The photograph is comparable to thebinary image of FIG. 1B.

FIG. 3 is a photograph of a human metaphase spread prepared andhybridized contemporaneously with the spreads shown in FIGS. 1A, B and Cand 2A and B. The procedures used were the same except that PI(propidium iodide) instead of DAPI, was used to stain all thechromosomes. Both PI and fluorescein stains can be viewed with the samemicroscope filters. Color film was used such that the propidium iodidecounterstain appears red and the fluorescein of the probe appears yellowon the color film.

FIG. 4A shows the hybridization of the chromosome 4-specific library inBluescribe plasmids (the library pBS-4) to a human metaphase spreadwherein no unlabeled human genomic DNA was used, and wherein thehybridization mixture was applied immediately after denaturation. Bothcopies of chromosome 4 are seen as slightly brighter than the otherchromosomes. The small arrows indicate regions that are unstained withthe probe. As in FIG. 3 and as in the rest of the FIGS. below, PI is thecounterstain and fluorescein is used to label the probe.

FIG. 4B shows the hybridization of pBS-4 to a human metaphase spreadwherein unlabeled human genomic DNA was used during the hybridization(Q=2 of genomic DNA; the meaning of Q is explained infra). Quantitativeimage analysis shows that the intensity per unit length of the chomosome4s is about 20X that of the other chromosomes. The chromosome 4s areyellow; the other chromosomes are red due to the propidium iodidecounterstain. Two layers of avidin-fluorescein isothiocyanate have beenused to make the target chromosomes sufficiently bright to be measuredaccurately. However, the number 4 chromosomes can be recognized easilyafter a single layer is applied.

FIG. 4C shows the same spread as in FIG. 4B but through a filter thatpasses only the fluorescein isothiocyanate fluorescence.

FIG. 4D shows the detection of a radiation-induced translocation(arrows) involving chromosome 4s in a human metaphase spread whereinpBS-4 specific libraries are used. The contrast ratio is about 5X.

FIG. 4E shows that normal and two derivative chromosomes resulting froma translocation between chromosome 4 and 11 (in cell line RS4;11) can bedetected by the compositions and methods of this invention in interphasenuclei. They appear as three distinct domains.

FIG. 4F shows the hybridization of the chromosome 21-specific library inBluescribe plasmids (the library pBS-21) to a metaphase spread of atrisomy 21 cell line. A small amount of hybridization is visible nearthe centromeres of the other acrocentric chromosomes.

FIG. 4G shows the same hybridization as in FIG. 4F but with interphasenuclei. Clearly shown are the three chromosome 21 domains.

FIG. 4H shows the hybridization with a pool of 120 single copy probesfrom chromosome 4 to a human metaphase spread. The number 4 chromosomesare indicated by arrows.

FIG. 5 shows the hybridization of a yeast artificial chromosome (YAC)clone containing a 580 kb insert of human DNA to a human metaphasespread. A yellow fluorescein band on each of the chromosome 12s (at12q21.1) is visible against the propidium iodide counterstain.

FIG. 6 shows the hybridization of DNA from a human/hamster hybrid cellcontaining on e copy of human chromosome 19 to a human metaphase spread.A little to the right of the photograph's center are the two chromosome19s which are brighter than the other chromosomes in the spread.

FIG. 7 illustrates a representative method of using the polymerase chainreaction (PCR) to produce probes of this invention which are reduced inrepetitive sequence.

FIG. 8 illustrates the locations of probes to the CML breakpoint andcorresponding pattern of staining in both normal and CML metaphase andinterphase nuclei.

The left side shows schematic representations of the BCR gene onchromosome 22, the ABL gene of chromosome 9, and the BCR-ABL fusion geneon the Philadelphia chromosome. Also show n are the locations of CMLbreakpoints and their relation to the probes (32). The right showshybridization patterns expected for the c-hu-ABL and PEM12 probes tonormal and CML metaphase spreads and interphase nuclei.

FIG. 9 shows fluorescence in-situ hybridization (FISH) in metaphasespreads and interphase nuclei. Panels A and B show ABL and BCRhybridization to normal metaphase spreads. The ABL signal (A) islocalized to the telomeric portion of 9q and the BCR signal (B) islocalized near the centromere of 22q. Panel C shows that abl staining islocalized to the telomeric region of Philadelphia chromosome in a caseof CML with 46XY, t (9:22) (q34;q11). Panel D shows that abl staining isinterstitial on the derivative 22 chromosome arising from an insertionalevent in a case of CML with 46XY ins (22:9)(q11;q34). Panel Eillustrates that the K562 cell line presents multiple signals localizedto a region of the interphase nucleus. Identical staining pattern wasseen with BCR probe indicating BCR-ABL fusion gene amplification. PanelF presents a metaphase spread from the K562 cell line showing fusiongene amplification localized to a single chromosome.

FIG. 10 illustrates fluorescence in-situ hybridization in CML interphasenuclei with ABL (red) and BCR (green) probes visualized simultaneouslythrough a double band pass filter. Cells from a CML patient show thered-green (yellow) signals resulting from the hybridization to theBCR-ABL fusion gene and single red and green hybridization signals tothe normal BCR and ABL genes on chromosomes 22 and 9.

FIG. 11 illustrates some exemplary probe strategies for detection ofstructural aberrations. The design of the binding pattern, colors etc.,of the probe can be optimized for detection of genetic abnormalities inmetaphase and/or interphase cells. Different patterns may haveadvantages for particular applications. The drawings in FIG. 11illustrate some of the patterns useful for detection of someabnormalities. The examples are representative and not meant to beexhaustive; different patterns can be combined to allow for thedetection of multiple abnormalities in the same cell.

In the drawings of FIG. 11, the metaphase chromosomes are shown withprobe bound to both chromatids. The interphase nuclei are pictured to bein a stage of the cell cycle prior to replication of the portion of thechromosome to which the probe binds; thus there is only one chromatidfor each interphase chromosome. When the probe binding is restricted toonly a portion of a chromosome, the signal is indicated as either ablack or white circle. Such a representation is employed to indicatedifferent colors or otherwise distinguishable characteristics of thestaining. Patterns containing more than two distinguishablecharacteristics (three colors, different ratios of colors etc.) permitmore complex staining patterns than those illustrated. Chromosomallocations of the breakpoints in the DNA are indicated with horizontallines next to the abnormal chromosomes.

a. Section (a) represents the use of a probe which stains a wholechromosome. Such a probe can be used to detect a translocation thatoccurs anywhere along the chromosome. The color photograph of FIG. 12shows use of such a stain for chromosome 22 to detect a translocation,in this case that which occurs with CML. Such an approach to staining isnot very useful in interphase nuclei since the region of the nucleusthat is stained is relatively large; overlaps in the stained regions canmake interpretation difficult in many nuclei.

b. Section (b) represents the reduction of the stained region of thechromosome shown in a) to that in the vicinity of a breakpoint,providing information focused on events in that region. The stainingpattern can be continuous or discontinuous across the breakpoint, justso that some binding is on both sides of the breakpoint. Such a stainingpattern requires only one “color”, but gives no information about whichother genomic region may be involved in the exchange.

c. Section (c) represents the use of a probe which binds to sequenceswhich come together as a result of the rearrangement and allows for thedetection in metaphase and interphase cells. In this case the differentsequences are stained with different “colors”. Such a staining patternis that used in the examples of Section VIII of the this application.

d. Section (d) represents an extension of (c) by including staining ofboth sides of both breakpoints involved in the rearrangement. Different“colors” are used as indicated. The additional information supplied bythe more complex staining pattern may assist with interpretation of thenuclei. It might also permit recognition of an apparent insertionalevent as discussed herein.

e. Section (e) represents the detection of an inversion in one homologueof a chromosome.

f. Section (f) represents a staining pattern useful in the detection ofa deletion. A deletion could also be detected with a probe that stainsonly the deleted region; however, lack of probe binding may be due toreasons other than deletion of the target sequence. The flanking regionsstained a different “color” serve as controls for hybridization.

FIG. 12 illustrates a staining pattern to detect a rearrangement bystaining a whole chromosome, in this case a rearrangement of chromosome22 associated with CML. The metaphase spread of this figure is from aCML cell that has been stained with a probe which binds all alongchromosome 22. Probe-stained regions appear yellow. The rest of the DNAhas been stained with the red-fluorescing chemical stain propidiumiodide. The entirely yellow chromosome is a normal copy of chromosome22. Just below said normal chromosome 22 is the Philadelphia chromosome,a small part yellow and part red chromosome. Below and to the right ofthe Philadelphia chromosome is the abnormal chromosome 9 (red) with thedistal part of chromosome 22 (yellow) attached. The photograph of thisfigure illustrates the staining pattern represented in part a) of theprevious figure.

FIG. 13 shows FISH with fourteen Rb-1 lambda phage clones (Rb-1 probe)in normal and abnormal metaphase spreads and interphase nuclei Panels Aand B show two pairs of bright and specific hybridization signals onnormal lymphocyte metaphase preparations in the mid-region of the q-armof chromosome 13. Panel B further shows cohybridization with a 13/21centromeric probe. Panel C shows a digital image analysis of the mappingof the Rb-1 gene on a metaphase chromosome using both the Rb-1 probe andthe 13/21 centromeric-specific repeat probe. Panel D shows two brightand specific hybridization domains in interphase nuclei of normallymphocytes. Panel E shows cohybridization of the Rb-i probe and a 13/21centromeric-specific repeat probe to metaphase spreads of a fibroblastcell line (GM05877) derived from a sporadic retinoblastoma patient.Intact chromosome 13s show both Rb-1 and centromere signals; whereaschromosome 13s with a Rb-1 deletion are slightly shortened and hybridizeonly with the centromeric probe. Panel F shows a digital image analysisof the GM05887 cell line metaphase showing both the normal and shortenedchromosome 13 and wherein cohybridization was effected with both theRb-1 and 13/21 centromeric probe. Panel G shows hybridization of theRb-1 probe to a GM05887 cell line interphase. Panel H showshybridization of the Rb-1 probe to a clinical breast cancer specimen.Panel I shows a digital image analysis of a dual color hybridization toa normal interphase nucleus; differently labeled protions of the Rb-1probe—a 3′(green) portion and a 5′ (red) portion—were hybridized to thenormal interphase nucleus.

FIG. 14 graphically illustrates the distribution of Rb-1 genehybridization signals in (A) interphase nuclei of normal peripheralblood lymphocytes and fibroblasts; and in (B) two cell lines with acytogenetically defined deletion involving the Rb-1 gene at 13q. Theresults represent the mean (+S.D.) of 3-5 different hybridizationexperiments. At least 150 nuclei were scored from each slide.

FIG. 15 shows FISH with chromosome 17 centromeric-specific alphasatellite probe. Panel A shows such hybridization to normal lymphocyteswherein in metaphase chromosomes, two chromosome 17 centromere-specificbright signals are seen and in interphase nuclei, corresponding brightand tight hybridization domains are visible. Panel B shows suchhybridization to an ovarian cancer cell line (RMUG-L) wherein in bothmetaphase and interphase, four signals are visible, indicatinganeuploidy of chromosome 17.

FIG. 16 shows FISH with a whole chromosome composite probe forchromosome 3. Panel A shows such a hybridization to normal lymphocyteswherein the whole body of chromosome 3 was homogeneously painted withfluorescein isothiocyanate (FITC). Panel B shows such a hybridization toan ovarian cancer cell line (RMUG-L) wherein four chromosome 3s arespecifically stained with FITC and wherein two (to which short arrowspoint) out of the four are apparently shorter than the intact chromosome3s (to which long arrows point). The shorter chromosomes are consideredto correspond to those with a 3p deletion.

FIG. 17 shows simultaneous hybridization with a chromosome 3centromeric-specific probe generated by the polymerase chain reaction(PCR) and a chromosome 3 locus-specific cosmid probe (mapped to ch. 3q26by digital image analysis). Panel A shows such a hybridization tometaphase spreads and interphase nuclei from normal lymphocytes whereintwo chromosome 3 centromeric-specific signals (indicated by shortarrows) and two pairs of chromosome 3q cosmid signals (indicated by longarrows) are clearly visible in the metaphase spreads; and wherein twolarge hybridization domains for the chromosome 3 centromere and twosmall domains for the chromosome 3q locus-specific probe are visible inthe interphase nuclei. Panel B shows such a hybridization to a uterinecervical adenocarcinoma cell line (TMCC-1) wherein two chromosome 3centromere-specific (indicated by short arrows) and two chromosome 3qlocus-specific cosmid (indicated by long arrows) signals are clearlyvisible in metaphase spreads whereas a pair of cosmid signals specificto chromosome 3q are found to be translocated to another chromosome.

FIG. 18 shows the simultaneous dual color hybridization with achromosome 3 centromeric-specific probe (green) and a chromosome 3locus-specific cosmid probe mapped to 3p21 (red) to (A) metaphasespreads and (B) interphase nuclei of normal lymphocytes.

FIG. 19 shows hybridizations corresponding to those shown in FIG. 18wherein the metaphase spreads (A) and interphase nucleus (B) are from anovarian cancer cell line (RMUG-S). In Panel A, the metaphase chromosomeon the right exhibits an apparent 3p deletion whereas the metaphasechromosome on the left appears intact. In Panel B, chromosome 3aneuploidy is demonstrated by the four green centromeric domains; twointact chromosome 3s are indicated by two pairs of adjacent green andred dots; and two 3p deleted chromosome 3s are indicated by the twosingle green domains.

FIG. 20 shows the simultaneous hybridizations of an AAF-labeledchromosome 3 centromere-specific probe (from H. Willard at Stanford) anda biotinylated chromosome 3q cosmid probe (J14R1A12; probes describedinfra under Section X) to a metaphase spread and interphase nucleus ofnormal lymphocytes. A normal pattern is shown, that is, two green andtwo pairs of red signals per cell.

FIG. 21 shows hybridizations comparable to that shown in FIG. 20 exceptthat the interphase nucleus is from an ovarian cancer cell line(RMUG-S). An abnormal pattern is shown, that is, four chromosome 3centromere-specific green signals and four chromosome 3q cosmid redsignals, indicating that the nucleus contains four long arms ofchromosome 3.

DETAILED DESCRIPTION OF THE INVENTION

This invention concerns the use of nucleic acid probes to stain targetedchromosomal material in patterns which can extend along one or morewhole chromosomes, and/or along one or more regions on one or morechromosomes, including patterns which extend over an entire genome. Thestaining reagents of this invention facilitate the microscopic and/orflow cytometric identification of normal and aberrant chromosomes andprovide for the characterization of the genetic nature of particularabnormalities, such as, genetic rearrangements. The term“chromosome-specific” is herein defined to encompass the terms “targetspecific” and “region specific”, that is, when the staining compositionis directed to one chromosome, it is chromosome-specific, but it is alsochromosome-specific when it is directed, for example, to multipleregions on multiple chromosomes, or to a region of only one chromosome,or to regions across the entire genome. The term chromosome-specificoriginated from the use of recombinant DNA libraries made by cloning DNAfrom a single normal chromosome type as the source material for theinitial probes of this invention. Libraries made from DNA from regionsof one or more chromosomes are sources of DNA for probes for that regionor those regions of the genome. The probes produced from such sourcematerial are region-specific probes but are also encompassed within thebroader phrase “chromosome-specific” probes. The term “target specific”is interchangeably used herein with the term “chromosome-specific”.

The word “specific” as commonly used in the art has two somewhatdifferent meanings. The practice is followed herein. “Specific” mayrefer to the origin of a nudeic acid sequence or to the pattern withwhich it will hybridize to a genome as part of a staining reagent. Forexample, isolation and cloning of DNA from a specified chromosomeresults in a “chromosome-specific library”. [Eg., Van Dilla et al.,“Human Chromosome-Specific DNA Libraries: Construction andAvailability,” Biotechnology, 4:537 (1986).] However, such a librarycontains sequences that are shared with other chromosomes. Such sharedsequences are not chromosome-specific to the chromosome from which theywere derived in their hybridization properties since they will bind tomore than the chromosome of origin. A sequence is “chromosome-specific”if it binds only to the desired portion of a genome. Such sequencesinclude single-copy sequences contained in the target or repetitivesequences, in which the copies are contained predominantly in thetarget.

“Chromosome-specific” in modifying “staining reagent” refers to theoverall hybridization pattern of the nucleic acid sequences thatcomprise the reagent. A staining reagent is chromosome-specific ifuseful contrast between the target and non-target chromosomal materialis achieved (that is, that the target can be adequately visualized).

A probe is herein defined to be a collection of nucleic acid fragmentswhose hybridization to the target can be detected. The probe is labeledas described below so that its binding to the target can be visualized.The probe is produced from some source of nucleic acid sequences, forexample, a collection of clones or a collection of polymerase chainreaction (PCR) products. The source nucleic add may then be processed insome way, for example, by removal of repetitive sequences or blockingthem with unlabeled nucleic add with complementary sequence, so thathybridization with the resulting probe produces staining of sufficientcontrast on the target. Thus, the word probe may be used herein to refernot only to the detectable nucleic acid, but also to the detectablenucleic acid in the form in which it is applied to the target, forexample, with the blocking nucleic acid, etc. The blocking nucleic acidmay also be mentioned separately. What “probe” refers to specificallyshould be clear from the context in which the word is used.

When two or more nucleic acid probes of this invention are mixedtogether, they produce a new probe which when hybridized to a targetaccording to the methods of this invention, produces a staining patternthat is a combination of the staining patterns individually produced bythe component probes thereof. Thus, the terms “probe” and “probes” (thatis, the singular and plural forms) can be used interchangeably withinthe context of a staining pattern produced. For example, if one probe ofthis invention produces a dot on chromosome 9, and another probeproduces a band on chromosome 11, together the two probes form a probewhich produces a dot/band staining pattern.

The term “labeled” is herein used to indicate that there is some methodto visualize the bound probe, whether or not the probe directly carriessome modified constituent. Section III infra describes various means ofdirectly labeling the probe and other labeling means by which the boundprobe can be detected.

The terms “staining” or “painting” are herein defined to meanhybridizing a probe of this invention to a genome or segment thereof,such that the probe reliably binds to the targeted chromosomal materialtherein and the bound probe is capable of being visualized. The terms“staining” or “painting” are used interchangeably. The patternsresulting from “staining” or “painting” are useful for cytogeneticanalysis, more particularly, molecular cytogenetic analysis. Thestaining patterns facilitate the microscopic and/or flow cytometricidentification of normal and abnormal chromosomes and thecharacterization of the genetic nature of particular abnormalities.Section III infra describes methods of rendering the probe visible.Since multiple compatible methods of probe visualization are available,the binding patterns of different components of the probe can bedistinguished—for example, by color. Thus, this invention is capable ofproducing any desired staining pattern on the chromosomes visualizedwith one or more colors (a multi-color staining pattern) and/or otherindicator methods. The term “staining” as defined herein does notinclude the concept of staining chromosomes with chemicals as inconventional karotyping methods although such conventional stains may beused in conjunction with the probes of this invention to allowvisualization of those parts of the genome where the probe does notbind. The use of DAPI and propidium iodide for such a purpose isillustrated in the figures.

The phrase “high complexity” is defined herein to mean that the probe,thereby modified contains on the order of 50,000 (50 kb) or greater, upto many millions or several billions, of bases of nucleic acid sequenceswhich are not repeated in the probe. For example, representative highcomplexity nucleic acid probes of this invention can have a complexitygreater than 50 kb, greater than 100,000 bases (100 kb), greater than200,000 (200 kb), greater than 500,000 bases (500 kb), greater than onemillion bases (1 Mb), greater than 2 Mb, greater than 10 Mb, greaterthan 100 Mb, greater than 500 Mb, greater than 1 billion bases and stillfurther greater than several billion bases.

The term “complexity” is defined herein according to the standard fornucleic acid complexity as established by Britten et al., Methods ofEnzymol., 29:363 (1974). See also Cantor and Schimmel, BiophysicalChemistry: Part III: The Behavior of Biological Macromolecules, at1228-1230 (Freeman and Co. 1980) for further explanation andexemplification of nucleic acid complexity.

The complexity preferred for a probe composition of this invention isdependent upon the application for which it is designed. In general, thelarger the target area, the more complex is the probe. It is anticipatedthat the complexity of a probe needed to produce a desired pattern oflandmarks on a chromosome will decrease as hybridization sensitivityincreases, as progress is made in hybridization technology. As thesensitivity increases, the reliability of the signal from smaller targetsites will increase. Therefore, whereas from about a 40 kb to about a100 kb target sequence may be presently necessary to provide a reliable,easily detectable signal, smaller target sequences should providereliable signals in the future. Therefore, as hybridization sensitivityincreases, a probe of a certain complexity, for example, 100 kb, shouldenable the user to detect considerably more loci in a genome than arepresently reliably detected; thus, more information will be obtainedwith a probe of the same complexity. The term “complexity” thereforerefers to the complexity of the total probe no matter how many visuallydistinct loci are to be detected, that is, regardless of thedistribution of the target sites over the genome.

As indicated above, with current hybridization techniques it is possibleto obtain a reliable, easily detectable signal with a probe of about 40kb to about 100 kb (eg. the probe insert capacity of one or a fewcosmids) targeted to a compact point in the genome. Thus, for example, acomplexity in the range of approximately 100 kb now permitshybridization to both sides of a tumor-specific translocation. Theportion of the probe targeted to one side of the breakpoint can belabeled differently from that targeted to the other side of thebreakpoint so that the two sides can be differentiated with differentcolors, for example. Proportionately increasing the complexity of theprobe permits analysis of multiple compact regions of the genomesimultaneously. The conventional banding patterns produced by chemicalstains may be replaced according to this invention with a series ofprobe-based, color coded (for example), reference points along eachchromosome or significant regions thereof.

Uniform staining of an extended contiguous region of a genome, forexample, a whole chromosome, requires a probe complexity proportional tobut substantially less than, the complexity of the target region. Thecomplexity required is only that necessary to provide a reliable,substantially uniform signal on the target. Section V.B, infra,demonstrates that fluorescent staining of human chromosome 21, whichcontains about 50 megabases (Mb) of DNA, is sufficient with a probecomplexity of about 1 Mb. FIG. 4H illustrates hybridization of about 400kb of probe to human chromosome 4, which contains about 200 Mb of DNA.In that case, gaps between the hybridization of individual elements ofthe probe are visible. FIGS. 4B and 4F demonstrate the results achievedwith probes made up of entire libraries for chromosomes 4 and 21,respectively. The chromosomes are stained much more densely as shown inFIGS. 4B and 4F than with the lower complexity probe comprisingsingle-copy nucleic acid sequences used to produce the pattern of FIG.4H.

Increasing the complexity beyond the minimum required for adequatestaining is not detrimental as long as the total nucleic acidconcentration in the probe remains below the point where hybridizationis impaired. The decrease in concentration of a portion of a sequence inthe probe is compensated for by the increase in the number of targetsites. In fact, when using double-stranded probes, it is preferred tomaintain a relatively low concentration of each portion of sequence toinhibit reassociation before said portion of sequence can find a bindingsite in the target.

The staining patterns of this invention comprise one or more “bands”.The term “band” is herein defined as a reference point in a genome whichcomprises a target nucleic acid sequence bound to a probe component,which duplex is detectable by some indicator means, and which at itsnarrowest dimension provides for a reliable signal under the conditionsand protocols of the hybridization and the instrumentation, among othervariables, used. A band can extend from the narrow dimension of asequence providing a reliable signal to a whole chromosome to multipleregions on a number of chromosomes.

The probe-produced bands of this invention are to be distinguished frombands produced by chemical staining as indicated above in theBackground. The probe-produced bands of this invention are based uponnudeic acid sequence whereas the bands produced by chemical stainingdepend on natural characteristics of the chromosomes, but not the actualnucleic add sequence. Further, the banding patterns produced by chemicalstaining are only interpretable in terms of metaphase chromosomeswhereas the probe-produced bands of this invention are useful both formetaphase and interphase chromosomes.

One method of forming the probes of the present invention is to poolmany different low complexity probes. Such a probe would then comprise a“heterogeneous mixture” of individual cloned sequences. The number ofclones required depends on the extent of the target area and thecapacity of the cloning vector. If the target is made up of severaldiscrete, compact loci, that is, single spots at the limit ofmicroscopic resolution, then about 40 kb, more preferably 100 kb, foreach spot gives a reliable signal given current techniques. The portionof the probe for each spot may be made up from, for example, a singleinsert from a yeast artificial chromosome (YAC), from several cosmidseach containing 35-40 kb or probe sequence, or from about 25 plasmidseach with 4 kb of sequence.

Representative heterogeneous mixtures of clones exemplified hereininclude phage (FIGS. 1, 2 and 3), and plasmids (FIG. 4). Yeastartificial chromosomes (YACS) (FIG. 5), and a single human chromosome inan inter-species hybrid cell (FIG. 6) are examples of high complexityprobes for single loci and an entire chromosome that can be propagatedas a single clone.

A base sequence at any point in the genome can be classified as either“single-copy” or “repetitive”. For practical purposes the sequence needsto be long enough so that a complementary probe sequence can form astable hybrid with the target sequence under the hybridizationconditions being used. Such a length is typically in the range ofseveral tens to hundreds of nucleotides.

A “single-copy sequence” is that wherein only one copy of the targetnucleic acid sequence is present in the haploid genome. “Single-copysequences” are also known in the art as “unique sequences”. A“repetitive sequence” is that wherein there are more than one copy ofthe same target nudeic acid sequence in the genome. Each copy of arepetitive sequence need not be identical to all the others. Theimportant feature is that the sequence be sufficiently similar to theother members of the family of repetitive sequences such that under thehybridization conditions being used, the same fragment of probe nucleicacid is capable of forming stable hybrids with each copy. A “sharedrepetitive sequence” is a sequence with some copies in the target regionof the genome, and some elsewhere.

When the adjectives “single-copy”, “repetitive”, “shared repetitive”,among other such modifiers, are used to describe sequences in the probe,they refer to the type of sequence in the target to which the probesequence will bind. Thus, “a repetitive probe” is one that binds to arepetitive sequence in the target; and “a single-copy probe” binds to asingle-copy target sequence.

Repetitive sequences occur in multiple copies in the haploid genome. Thenumber of copies can range from two to hundreds of thousands, whereinthe Alu family of repetitive DNA are exemplary of the latter numerousvariety. The copies of a repeat may be clustered or interspersedthroughout the genome. Repeats may be clustered in one or more locationsin the genome, for example, repetitive sequences occurring near thecentromeres of each chromosome, and variable number tandem repeats(VNTRs) [Nakamura et al, Science, 235:1616 (1987)]; or the repeats maybe distributed over a single chromosome [for example, repeats found onlyon the X chromosome as described by Bardoni et al., Cytogenet. CellGenet., 46:575 (1987)]; or the repeats may be distributed over all thechromosomes, for example, the Alu family of repetitive sequences.

Herein, the terms repetitive sequences, repeated sequences and repeatsare used interchangeably.

Shared repetitive sequences can be clustered or interspersed. Clusteredrepetitive sequences include tandem repeats which are so named becausethey are contiguous on the DNA molecule which forms the backbone of achromosome. Clustered repeats are associated with well-defined regionsof one or more chromosomes, e.g., the centromeric region. If one or moreclustered repeats form a sizable fraction of a chromosome, and areshared with one or more non-target regions of the genome and areconsequently removed from the heterogeneous mixture of fragmentsemployed in the invention or the hybridization capacity thereof isdisabled, perfect uniformity of staining of the target region may not bepossible. That situation is comprehended by the use of the term“substantially uniform” in reference to the binding of the heterogeneousmixture of labeled nucleic acid fragments to the target.

Chromosome-specific staining of the current invention is accomplished byusing nucleic acid fragments that hybridize to sequences specific to thetarget. These sequences may be either single-copy or repetitive, whereinthe copies of the repeat occur predominantly in the target area. FIG. 4Hand the results of the work detailed in section V infra indicate thatprobes can be made of single-copy sequences. However, in probes such asthat of FIG. 4B, low-copy chromosome-specific repeats [Nakamura et al.,and Bardoni et al., supra] may contribute to the hybridization as well.

If nucleic acid fragments complementary to non-target regions of thegenome are included in the probe, for example, shared repetitivesequences or non-specific sequences, their hybridization capacity needsto be sufficiently disabled or their prevalence sufficiently reduced, sothat adequate staining contrast can be obtained. Section V and FIG. 4Hshow examples of hybridization with probes that contain pools of clonesin which each clone has been individually selected so that it hybridizesto single-copy sequences or very low copy repetitive sequences. Theremaining figures illustrate use of probes that contain fragments thatcould have hybridized to high-copy repetitive sequences, but which havehad the hybridization capacity of such sequences disabled.

The nucleic acid probes of this invention need not be absolutelyspecific for the targeted portion of the genome. They are intended toproduce “staining contrast”. “Contrast” is quantified by the ratio ofthe stain intensity of the target region of the genome to that of theother portions of the genome. For example, a DNA library produced bycloning a particular chromosome, such as those listed in Table I, can beused as a probe capable of staining the entire chromosome. The librarycontains sequences found only on that chromosome, and sequences sharedwith other chromosomes. In a simplified (approximately true to life)model of the human genome, about half of the chromosomal DNA falls intoeach class. If hybridization with the whole library were capable ofsaturating all of the binding sites, the target chromosome would betwice as bright (contrast ratio of 2) as the others since it wouldcontain signal from the specific and shared sequences in the probe,whereas the other chromosome would only have signal from the sharedsequences. Thus, only a modest decrease in hybridization of the sharedsequences in the probe would substantially enhance the contrast.Contaminating sequences which only hybridize to non-targeted sequences,for example, impurities in a library, can be tolerated in the probe tothe extent that said sequences do not reduce the staining contrast belowuseful levels.

In reality all of the target sites may not be saturated during thehybridization, and many other mechanisms contribute to producingstaining contrast, but this model illustrates one general considerationin using probes targeted at a large portion of a genome.

The required contrast depends on the application for which the probe isdesigned. When visualizing chromosomes and nuclei, etc.,microscopically, a contrast ratio of two or greater is often sufficientfor identifying whole chromosomes. In FIGS. 4D-F, the contrast ratio is3-5. The smaller the individual segments of the target region, thegreater the contrast needs to be to permit reliable recognition of thetarget relative to the fluctuations in staining of the non-targetedregions. When quantifying the amount of target region present in a cellnucleus by fluorescence intensity measurements using flow cytometry orquantitative microscopy, the required contrast ratio is on the order of1/T or greater on average for the genome, where T is the fraction of thegenome contained in the targeted region. When the contrast ratio isequal to 1/T, half of the total fluorescence intensity comes from thetarget region and half from the rest of the genome. For example, whenusing a high complexity probe for chromosome 1, which comprises about10% of the genome, the required contrast ratio is on the order of 10,that is, for the chromosome 1 fluorescence intensity to equal that ofthe rest of the genome.

Background staining by the probe, that is, to the non-target region ofthe genome, may not be uniform. FIG. 4F shows that a chromosome 21specific probe contains probe fragments that hybridize weakly to compactregions near the centromeres of other acrocentric human chromosomes.This degree of non-specificity does not inhibit its use in theillustrated applications. For other applications, removal of or furtherdisabling the hybridization capacity of the probe fragments that bind tothese sequences may be necessary.

For other applications, repetitive sequences that bind to centromeres,for example, alpha-satellite sequences, and/or telomeres can be part ofthe chromosome-specific staining reagents wherein the target includessome or all of the centromeres and/or telomeres in a genome along withperhaps other chromosomal regions. Exemplary of such an applicationwould be that wherein the staining reagent is designed to detect randomstructural aberrations caused by clastogenic agents that result indicentric chromosomes and other structural abnormalities, such astranslocations. Addition of sequences which bind to all centromeres in agenome, for example to the probe used to create the staining pattern ofFIG. 4D, would allow more reliable distinguishing between dicentrics andtranslocations.

Application of staining reagents of this invention to a genome resultsin a substantially uniform distribution of probe hybridized to thetargeted regions of a genome. The distribution of bound probe is deemed“substantially uniform” if the targeted regions of the genome can bevisualized with useful contrast. For example, a target is substantiallyuniformly stained in the case wherein it is a series of visuallyseparated loci if most of the loci are visible in most of the cells.

“Substantial proportions” in reference to the base sequences of nucleicacid fragments that are complementary to chromosomal DNA means that thecomplementarity is extensive enough so that the fragments form stablehybrids with the chromosomal DNA under the hybridization conditionsused. In particular, the term comprehends the situation where thenucleic acid fragments of the heterogeneous mixture possess some regionsof sequence that are not perfectly complementary to target chromosomalmaterial. The stringency can be adjusted to control the precision of thecomplementarity required for hybridization.

The phrase “metaphase chromosomes” is herein defined to mean not onlychromosomes condensed in the metaphase stage of mitosis but includes anycondensed chromosomes, for example, those condensed by prematurechromosome condensation.

To disable the hybridization capacity of a nudeic acid sequence isherein sometimes abbreviated as “disabling the nucleic acid sequence”.

The methods and reagents of this invention find a particularlyappropriate application in the field of diagnostic cytogenetics,particularly in the field of diagnostic interphase cytogenetics.Detecting genetic rearrangements that are associated with a disease,such as cancer, are a specific application of the chromosome specificreagents and staining methods of this invention.

Contiguous gene syndromes are an example of the genetic rearrangementsthat the probes and methods of this invention can identify. Contiguousgene syndromes are characterized by the presence of several closelyspaced genes which are in multiple and/or reduced copy number. Downsyndrome is an example of a contiguous gene syndrome wherein an extracopy of a chromosomal region containing several genes is present.

Particularly described herein is the application of chromosome specificreagents and methods for detecting genetic rearrangements that producethe BCR-ABL fusion associated with CML. Such reagents are exemplary ofdisease specific, in this case tumor specific, probes which can belabeled, directly and/or indirectly, such that they are visualizablewhen bound to the targeted chromosomal material, which in the case ofCML, is the vicinity of the translocation breakpoint regions ofchromosomal regions 9q34 and 22q11 known to be associated with CML. Inthe examples provided in Section VIII of this application, the probesare labeled such that a dual color fluorescence is produced in thestaining pattern of said probes upon in situ hybridization [fluorescentin situ hybridication (FISH)]; however, staining patterns can beproduced in many colors as well as other types of signals, and anyvisualization means to signal the probe bound to its target can be usedin the methods of this invention.

Section VIII herein describes representative methods and reagents ofthis invention to detect genetic rearrangements. The examples of SectionVIII concern genetic rearrangements that produce the BCR-ABL fusion thatis characteristic of CML. The approach in such examples is based on FISHwith probes from chromosomes 9 and 22 that flank the fused BCR and ABLsequences in essentially all cases of CML (FIG. 8). The probes whenhybridized to the chromosomal material of both normal and abnormal cellsproduce staining patterns that are different as illustrated in FIGS.8-12. The staining patterns produced by such exemplary probes aredifferent in normal and abnormal cells; the staining pattern presentwhen the genetic rearrangement occurs is distinctively altered from thatof the staining pattern shown by hybridizing the probes to chromosomalmaterial that does not contain the genetic rearrangement. Further,staining patterns are distinctively different for one type of geneticrearrangement versus another. For example, the staining patternsproduced upon hybridization of nucleic acid probes of this invention tochromosomal material containing a genetic rearrangement associated withALL is distinctively different from that produced upon hybridization ofsuch probes to chromosomal material containing the BCR-ABL fusioncharacteristic of CML. Thus, the methods and reagents of this inventionprovide for differential diagnosis of related diseases.

The examples of Section VIII provide for the diagnosis of CML based uponthe proximity of the fluorescent signals in the staining patterns, andrely upon a 1 micron cutoff point for determination of the presence of afusion. The proximity distance of signals is only one characteristic,among many others, of signals that can be used to detect the presence ofa genetic rearrangement. Further, the proximity distance is dependent onthe particular cell preparation techniques employed and the size of thenuclei therein, and for a particular cell preparation is relativedepending on the distance between signals in normal and abnormal cells.

The staining patterns exemplified in the examples of Section VIII arerepresentative of one type of probe strategy. Many other probestrategies can be employed. FIG. 11 illustrates some other exemplaryprobe strategies for detecting genetic rearrangements, the patterns ofwhich can be modified and optimized and otherwise varied to detectparticular genetic rearrangements.

Use of other disease specific reagents of this invention would beanalogous to the methods detailed in Section VIII for CML. For example,the diagnosis and study of acute lymphocytic leukemia (ALL) may beaccomplished by replacing the BCR probe (PEM12) of Section VIII with aprobe from the 5′ end of the BCR gene. ALL is of particular interestbecause the Ph′ chromosome is the most common cytogenetic abnormality inthat disease, and the presence of such a chromosome is indicative of avery aggressive neoplasm.

The methods and reagents herein exemplified, particularly in SectionVIII, provide for the means to distinguish between cytogeneticallysimilar but genetically different diseases. “Cytogenetically” in thatparticular context refers to a similarity determined by conventionalbanding analysis. CML and ALL are in that context cytogeneticallysimilar in that conventional banding analysis can not distinguish thembecause the breakpoints associated with each are so close together inthe human genome.

Further, this invention provides methods and reagents that can be usedin a cytogenetic research mode for the study of the molecular bases ofgenetic disease. For example, if an abnormality in a person's karyotypeis noted by conventional banding analysis, the probes and reagents ofthis invention can be used to detect any genetic rearrangements in thevicinity of said abnormality. The underlying molecular basis of theabnormality can be determined by the methods and reagents of thisinvention, and the resulting differences at the genetic level may beindicative of different treatment plans and prognostically important.The underlying genetic rearrangements may be found to be consistentlyassociated with a set of phenotypic characteristics in a population.

The following sections provide examples of making and using the stainingcompositions of this invention and are for purposes of illustration onlyand not meant to limit the invention in any way. The followingabbreviations are used.

Abbreviations

AAF—N-acetoxy-N-2-acetyl-aminofluorene

BN—bicarbonate buffer with NP-40

bp—base pair

CML—chronic myelogenous leukemia

DAPI—4,6-diamidino-2-phenylindole

dATP—deoxyadenosine triphosphate

DCS—as in fluorescein-avidin DCS (a commercially available cell sortergrade of fluorescein Avidin D)

dCTP—deoxycytosine triphosphate

dGTP—deoxyguanosine triphosphate

DI—DNA index

dNTP—deoxynucleotide triphosphate

dTTP—deoxythymidine triphosphate

dUTP—deoxyuridine triphosphate

EDTA—ethylenediaminetetraacetate

FISH—fluorescence in situ hybridization

FACS—fluorescence-activated cell sorting

FITC—fluorescein isothiocyanate

HPLC—high performance liquid chromatography

IB—isolation buffer

kb—kilobase

kDa—Kilodalton

NP-40—non-ionic detergent commercially available from Sigma as NonidetP-40 (St. Louis, Mo.)

PBS—phosphate-buffered saline

PHA—phytohemagglutinin

PCR—polymerase chain reaction

PI—propidium iodide

PMSF—phenylmethylsulfonyl fluoride

PN—mixture of 0.1 M NaH₂PO₄ and 0.1 M buffer Na₂HPO₄, pH 8; 0.1% NP-40

PNM—Pn buffer plus 5% nonfat dry milk (centrifuged); buffer 0.02% Naazide

Rb-1—retinoblastoma gene

RFLP—restriction fragment length polymorphism

SD—Standard Deviation

SDS—sodium dodecyl sulfate

SSC—0.15 M NaCl/0.015 M Na citrate, pH 7

VNTR—variable number tandem repeat

ug—microgram

ul—microliter

um—micrometer

uM—micromole

ml—milliliter

mM—milliMole

mm—millimeter

ng—nanogram

I. Methods of Preparing Chromosome-Specific Staining Reagents

I.A. Isolation of Chromosome-Specific DNA and Formation of DNA FragmentLibraries

The first step in a preferred method of making the compositions of theinvention is isolating chromosome-specific DNA (which term includestarget-specific and/or region-specific DNA, as indicated above, whereinspecific refers to the origin of the DNA). This step includes firstisolating a sufficient quantity of the particular chromosome type orchromosomal subregion to which the staining composition is directed,then extracting the DNA from the isolated chromosome(s) or chromosomalsubregion(s). Here “sufficient quantity” means sufficient for carryingout subsequent steps of the method. Preferably, the extracted DNA isused to create a library of DNA inserts by cloning using standardgenetic engineering techniques.

Preferred cloning vectors include, but are not limited to, yeastartificial chromosomes (YACS), plasmids, bacteriophages and cosmids.Preferred plasmids are Bluescribe plasmids; preferred bacteriophages arelambda insertion vectors, more preferably Charon 4A, Charon 21A, Charon35, Charon 40 and GEM11; and preferred cosmids include Lawrist 4,Lawrist 5 and sCos1.

As indicated above, the DNA can be isolated from any source.Chromosome-specific staining reagents can be made from both plant andanimal DNA according to the methods of this invention. Important sourcesof animal DNA are mammals, particularly primates or rodents whereinprimate sources are more particularly human and monkey, and rodentsources are more particularly rats or mice, and more particularly mice.

1. Isolating DNA from an Entire Chromosome. A preferred means forisolating particular whole chromosomes (specific chromosome types) is bydirect flow sorting [fluorescence-activated cell sorting (FACS)] ofmetaphase chromosomes with or without the use of interspecific hybridcell systems. For some species, every chromosome can be isolated bycurrently available sorting techniques. Most, but not all, humanchromosomes are currently isolatable by flow sorting from human cells,Carrano et al., “Measurement and Purification of Human Chromosomes byFlow Cytometry and Sorting,” Proc. Natl. Acad. Sci., Vol. 76, pgs.1382-1384 (1979). Thus, for isolation of some human chromosomes, use ofthe human/rodent hybrid cell system may be necessary, see Kao, “SomaticCell Genetics and Gene Mapping,” International Review of Cytology., Vol.85, pgs. 109-146 (1983), for a review, and Gusella et al., “Isolationand Localization of DNA Segments from Specific Human Chromosomes,” Proc.Natl. Acad. Sci., Vol. 77, pgs. 2829-2833 (1980). Chromosome sorting canbe done by commercially available fluorescence-activated sortingmachines, e.g., Becton Dickinson FACS-II, Coulter Epics V sorter, orspecial purpose sorters optimized for chromosome sorting or likeinstrument.

DNA is extracted from the isolated chromosomes by standard techniques,e.g., Marmur, “A Procedure for the Isolation of Deoxyribonucleic Acidfrom Micro-Organisms,” J. Mol. Biol., Vol. 3, pgs. 208-218 (1961); orManiatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, 1982) pgs. 280-281. These references are incorporatedby reference for their descriptions of DNA isolation techniques.

Generation of insert libraries from the isolated chromosome-specific DNAis carried out using standard genetic engineering techniques, e.g.,Davies et al., “Cloning of a Representative Genomic Library of the HumanX Chromosome After Sorting by Flow Cytometry,” Nature, Vol. 293, pgs.374-376 (1981); Krumlauf et al., “Construction and Characterization ofGenomic Libraries from Specific Human Chromosomes,” Proc. Natl. Acad.Sci., Vol. 79, pgs. 2971-2975 (1982); Lawn et al., “The Isolation andCharacterization of Linked Delta-and-Beta-Globin Genes from a ClonedLibrary of Human DNA.” Cell, Vol. 15, pgs. 1157-1174 (1978); andManiatis et al., “Molecular Cloning: A Laboratory Manual,” (Cold SpringsHarbor Laboratory, 1982), pgs. 256-308; Van Dilla et al., id; Fuscoe,Gene, 52:291 (1987); and Fuscoe et al., Cytogenet. Cell Genet., 43:79(1986). Said references are herein incorporated by reference.

Recombinant DNA libraries for each of the human chromosomes have beenconstructed by the National Laboratory Gene Library Project and areavailable from the American Type Culture Collection. [Van Dilla et al.,Biotechnology, 4:537 (1986).] Small insert-containing libraries wereconstructed by complete digestion of flow sorted human chromosomegenomic DNA with HindIII or EcoRI and cloning into the Lambda insertionvector Charon 21A. The vector is capable of accepting human inserts ofup to 9.1′ kb in size. Thus, HindIII (or EcoRI) restriction fragmentsgreater than 9.1′ kb will not be recovered from these libraries. Theobserved average insert size in these libraries is approximately 4 kb. Arepresentative list of the HindIII chromosome-specific libraries withtheir ATCC accession numbers are shown in Table 1.

TABLE 1 HUMAN CHROMOSOME - SPECIFIC GENOMIC LIBRARIES IN CHARON 21AVECTOR CHROMOSOME ATCC # LIBRARY 1 57753 LL01NS01 1 57754 LL01NS02 257744 LL02NS01 3 57751 LL03NS01 4 57700 LL04NS01 4 57745 LL04NS02 557746 LL05NS01 6 57701 LL06NS01 7 57755 LL07NS01 8 57702 LL08NS02 957703 LL09NS01 10 57736 LL10NS01 11 57704 LL11NS01 12 57756 LL12NS01 1357705 LL13NS01 13 57757 LL13NS02 14 57706 LL14NS01 14/15 57707 LL99NS0115 57737 LL15N5b1 16 57758 LL16NS03 17 57759 LL17NS02 18 57710 LL18NS0119 57711 LL19NS01 20 57712 LL20NS01 21 57713 LL21NS02 22 57714 LL22NS01X 57747 LL0XNS01 Y 57715 LL0YNS01

Alternatively, the extracted DNA from a sorted chromosome type can beamplified by the polymerase chain reaction (PCR) rather than cloning theextracted DNA in a vector or propagating it in a cell line. Appropriatetails are added to the extracted DNA in preparation for PCR. Referencesfor such PCR procedures are set out in Section I.B infra.

Other possible methods of isolating the desired sequences from hybridcells include those of Schmeckpeper et al., “Partial Purification andCharacterization of DNA from Human X Chromosome,” Proc. Natl. Acad. Sci,Vol. 76, pgs. 6525-6528 (1979); or Olsen et al., supra (in Background).Accordingly, these references are incorporated by reference.

2. Isolating DNA from a Portion of a Chromosome. Among the methods thatcan be used for isolating region-specific chromosomal DNA include theselection of an appropriate chromosomal region from DNA that haspreviously been mapped, for example, from a library of mapped cosmids;the sorting of derivative chromosomes, for example, by FACS; themicrodissection of selected chromosomal material; subtractivehybridization; identification of an appropriate hybrid cell containing adesired chromosomal fragment, extracting and amplifying the DNA, andselecting the desired amplified DNA; and the selection of appropriatechromosomal material from radiation hybrids. The standard geneticengineering techniques outlined above in subsection I.A.1 are used insuch procedures well-known to those in the art. Amplification of theregion-specific DNA can be performed by cloning in an appropriatevector, propagating in an appropriate cell line, and/or by the use ofPCR (see I.B infra).

A preferred method of isolating chromosomal region-specific DNA is touse mapped short DNA sequences to probe a library of longer DNAsequences, wherein the latter library has usually been cloned in adifferent vector. For example, a probe cloned in a plasmid can be usedto probe a cosmid or yeast artificial chromosome (YAC) library. By usingan initial seed probe, overlapping clones in the larger insert librarycan be found (a process called “walking”), and a higher complexity probecan be produced for reliable staining of the chromosomal regionsurrounding the seed probe. Ultimately, when an entire genome for aspecies has been mapped (for example, by the Human Genome Project forthe human species), ordered clones for the entire genome of the specieswill be available. One can then easily select the appropriate clones toform a probe of the desired specificity.

Another method of isolating DNA from a chromosomal region or regions (oralso a whole chromosome) is to propagate such a chromosomal region orregions in an appropriate cell line (for example, a hybrid cell linesuch as a human/hamster hybrid cell), extract the DNA from the cell lineand clone it in an appropriate vector and select clones containing humanDNA to form a library. When a hybrid cell is used, the chromosomes inthe hybrid cell containing the human chromosomal material may beseparated by flow sorting (FACS) prior to cloning to increase thefrequency of human clones in the library. Still further, total DNA fromthe hybrid cell can be isolated and labeled without further cloning andused as a probe, as exemplified in FIG. 6.

3. Single-Stranded Probes. In some cases, it is preferable that thenucleic acid fragments of the heterogeneous mixture consist ofsingle-stranded RNA or DNA. Under some conditions, the bindingefficiency of single-stranded nucleic acid probes has been found to behigher during in situ hybridization, e.g., Cox et al., “Detection ofmRNAs in Sea Urchin Embryos by In Situ Hybridization Using AsymmetricRNA Probes,” Developmental Biology, Vol. 101, pgs. 485-502 (1984).

Standard methods are used to generate RNA fragments from isolated DNAfragments. For example, a method developed by Green et al., described inCell, Vol. 32, pgs. 681-694 (1983), is commercially available fromPromega Biotec (Madison, Wis.) under the tradename “Riboprobe.” Othertranscription kits suitable for use with the present invention areavailable from United States Biochemical Corporation (Cleveland, Ohio)under the tradename “Genescribe.” Single-stranded DNA probes can beproduced with the single-stranded bacteriophage M13, also available inkit form, e.g. Bethesda Research Laboratories (Gaithersburg, Md.). Thehybridizations illustrated in FIG. 4 were performed with the librariesof Table 1 subcloned into the Bluescribe plasmid vector (Stratagene, LaJolla, Calif.). The Bluescribe plasmid contains RNA promoters whichpermit production of single-stranded probes.

Co-pending, commonly owned U.S. patent application No. 934,188 (filedNov. 24, 1986), entitled “Method of Preparing and Applying SingleStranded DNA Probes to Double Stranded Target DNAs,” provides methodsfor preparing and applying non-self-complementary single-strandednucleic acid probes that improve signal-to-noise ratios attainable insitu hybridization by reducing non-specific and mismatched binding ofthe probe. That application further provides for methods of denaturingdouble-stranded target nucleic acid which minimizes single-strandedregions available for hybridization that are non-complementary to probesequences. Said application is herein specifically incorporated byreference. Briefly, probe is constructed by treating DNA with arestriction enzyme and an exonuclease to form template/primers for a DNApolymerase. The digested strand is resynthesized in the presence oflabeled nudeoside triphosphate precursor, and the labeledsingle-stranded fragments are separated from the resynthesized fragmentsto form the probe. The target nudeic acid is treated with the samerestriction enzyme used to construct the probe, and is treated with anexonuclease before application of the probe.

I.B. PCR

Another method of producing probes of this invention includes the use ofthe polymerase chain reaction [PCR]. [For an explanation of themechanics of PCR, see Saiki et al., Science, 230:1350 (1985) and U.S.Pat. Nos. 4,683,195, 4,683,202 (both issued Jul. 28, 1987) and 4,800,159(issued Jan. 24, 1989).] Target-specific nucleic acid sequences,isolated as indicated above, can be amplified by PCR to producetarget-specific sequences which are reduced in or free of repetitivesequences. The PCR primers used for such a procedure are for the ends ofthe repetitive sequences, resulting in amplification of sequencesflanked by the repeats.

FIG. 7 illustrates such a method of using PCR wherein the representativerepetitive sequence is Alu. If only short segments are amplified, it isprobable that such sequences are free of other repeats, thus providingDNA reduced in repetitive sequences.

One can further suppress production of repetitive sequences in such aPCR procedure by first hybridizing complementary sequences to saidrepetitive sequence wherein said complementary sequences have extendednon-complementary flanking ends or are terminated in nucleotides whichdo not permit extension by the polymerase. The non-complementary ends ofthe blocking sequences prevent the blocking sequences from acting as aPCR primer during the PCR process.

II. Removal of Repetitive Sequences and/or Disabling the HybridizationCapacity of Repetitive Sequences

Typically a probe of the current invention is produced in a number ofsteps including: obtaining source nudeic acid sequences that arecomplementary to the target region of the genome, labeling and otherwiseprocessing them so that they will hybridize efficiently to the targetand can be detected after they bind, and treating them to either disablethe hybridization capacity or remove a sufficient proportion of sharedrepetitive sequences, or both disable and remove such sequences. Theorder of these steps depends on the specific procedures employed.

The following methods can be used to remove shared repetitive sequencesand/or disable the hybridization capacity of such shared repetitivesequences. Such methods are representative and are expressedschematically in terms of procedures well known to those of ordinaryskill the art, and which can be modified and extended according toparameters and procedures well known to those in the art.

1. Single-copy probes. A single-copy probe consists of nucleic acidfragments that are complementary to single-copy sequences contained inthe target region of the genome. One method of constructing such a probeis to start with a DNA library produced by cloning the target region.Some of the clones in the library will contain DNA whose entire sequenceis single-copy; others will contain repetitive sequences; and stillothers will have portions of single-copy and repetitive sequences.Selection, on a done by done basis, and pooling of those clonescontaining only single-copy sequences will result in a probe that willhybridize specifically to the target region. The single-copy nature of aclone can ultimately be established by Southern hybridization usingstandard techniques. FIG. 4H shows hybridization with 120 clonesselected in this way from a chromosome 4 library.

Southern analysis is very time consuming and labor intensive. Therefore,less perfect but more efficient screening methods for obtainingcandidate single-copy clones are useful. In Section V.B, examples ofimproved methods are provided for screening individual phage and plasmidclones for the presence of repetitive DNA using hybridization withgenomic DNA. The screening of plasmid clones is more efficient, andapproximately 80% of selected clones contain only single-copy sequences;the remainder contain low-copy repeats. However, probes produced in thisway can produce adequate staining contrast, indicating that the low-copyrepetitive sequences can be tolerated in the probe (see subsection 3 ofthis section).

A disadvantage of done by done procedures is that a done is discardedeven if only a portion of the sequence it contains is repetitive. Thelarger the length of the cloned nucleic acid, the greater the chancethat it will contain a repetitive sequence. Therefore, when nucleic acidis propagated in a vector that contains large inserts such as a cosmid,YAC, or in a cell line, such as hybrid cells, it may be advantageous tosubclone it in smaller pieces before the single-copy selection isperformed. The selection procedures just outlined above do notdiscriminate between shared and specific repetitive sequences; cloneswith detectable repetitive sequences of either type are not used in theprobe.

2. Individual testing of hybridization properties. The hybridizationspecificity of a piece of nucleic acid, for example, a clone, can betested by in situ hybridization. If under appropriate hybridizationconditions it binds to single-copy or repetitive sequences specific forthe desired target region, it can be included in the probe. Manysequences with specific hybridization characteristics are already known,such as chromosome-specific repetitive sequences [Trask et al., supra,(1988) and references therein], VNTRs, numerous mapped single copysequences. More are continuously being mapped. Such sequences can beincluded in a probe of this invention.

3. Bulk Procedures. In many genomes, such as the human genome, a majorportion of shared repetitive DNA is contained in a few families ofhighly repeated sequences such as Alu. A probe that is substantiallyfree of such high-copy repetitive sequences will produce useful stainingcontrast in many applications. Such a probe can be produced from somesource of nucleic acid sequences, for example, the libraries of Table I,with relatively simple bulk procedures. Therefore, such bulk proceduresare the preferred methods for such applications.

These methods primarily exploit the fact that the hybridization rate ofcomplementary nucleic acid strands increases as their concentrationincreases. Thus, if a heterogeneous mixture of nucleic acid fragments isdenatured and incubated under conditions that permit hybridization, thesequences present at high concentration will become double-stranded morerapidly than the others. The double-stranded nucleic acid can then beremoved and the remainder used as a probe. Alternatively, the partiallyhybridized mixture can be used as the probe, the double-strandedsequences being unable to bind to the target. The following are methodsrepresentative of bulk procedures that are useful for producing thetarget-specific staining of this invention.

3a. Self-reassociation of the probe. Double-stranded probe nucleic acidin the hybridization mixture is denatured and then incubated underhybridization conditions for a time sufficient for the high-copysequences in the probe to become substantially double-stranded. Thehybridization mixture is then applied to the sample. The remaininglabeled single-stranded copies of the highly repeated sequences bindthroughout the sample producing a weak, widely distributed signal. Thebinding of the multiplicity of low-copy sequences specific for thetarget region of the genome produce an easily distinguishable specificsignal.

Such a method is exemplified in Section VI.B (infra) withchromosome-specific libraries for chromosomes 4 and 21 (pBS4 and pBS21)as probes for those chromosomes. The hybridization mix, containing aprobe concentration in the range of 1-10 ng/ul was heated to denaturethe probe and incubated at 37° C. for 24 hours prior to application tothe sample.

3b. Use of blocking nucleic acid. Unlabeled nudeic acid sequences whichare complementary to those sequences in the probe whose hybridizationcapacity it is desired to inhibit are added to the hybridizationmixture. The probe and blocking nucleic acid are denatured, ifnecessary, and incubated under appropriate hybridization conditions. Thesequences to be blocked become double-stranded more rapidly than theothers, and therefore are unable to bind to the target when thehybridization mixture is applied to the target. In some cases, theblocking reaction occurs so quickly that the incubation period can bevery short, and adequate results can be obtained if the hybridizationmix is applied to the target immediately after denaturation. A blockingmethod is generally described by Sealy et al., “Removal of RepeatSequences form Hybridization Probes”, Nucleic Acid Research, 13:1905(1985), which reference is incorporated by reference. Examples ofblocking nudeic acids include genomic DNA, a high-copy fraction ofgenomic DNA and particular sequences as outlined below (i-iii).

3b.i. Genomic DNA. Genomic DNA contains all of the nudeic acid sequencesof the organism in proportion to their copy-number in the genome. Thus,adding genomic DNA to the hybridization mixture increases theconcentration of the high-copy repeat sequences more than low-copysequences, and therefore is more effective at blocking the former.However, the genomic DNA does contain copies of the sequences that arespecific to the target and so will also reduce the desiredchromosome-specific binding if too much is added. Guidelines todetermine how much genomic DNA to add (see 3.e. Concept of Q, infra) andexamples of using genomic blocking DNA are provided below. The blockingeffectiveness of genomic DNA can be enhanced under some conditions byadjusting the timing of its addition to the hybridization mix; examplesof such timing adjustments are provided with Protocol I and Protocol IIhybridizations illustrated in FIGS. 4B through E (Protocol I) and FIG.4F (Protocol II) and detailed in Section VI, infra.

3b.ii. High-copy fraction of genomic DNA. The difficulty with use ofgenomic DNA is that it also blocks the hybridization of the low-copysequences, which are predominantly the sequences that give the desiredtarget staining. Thus, fractionating the genomic DNA to obtain only thehigh-copy sequences and using them for blocking overcomes thisdifficulty. Such fractionation can be done, for example, withhydroxyapatite as described below (3c.i).

3b.iii. Specified sequences. The blocking of a particular sequence inthe probe can be accomplished by adding many unlabeled copies of thatsequence. For example, Alu sequences in the probe can be blocked byadding cloned Alu DNA. Blocking DNA made from a mixture of a few clonescontaining the highest copy sequences in the human genome can be usedeffectively with chromosome-specific libraries for example, those ofTable I. Alternatively, unlabeled nucleic acid sequences from one ormore chromosome-specific libraries could be used to block a probecontaining labeled sequences from one or more other chromosome-specificlibraries. The shared sequences would be blocked whereas sequencesoccurring only on the target chromosome would be unaffected. FIG. 4Fshows that genomic DNA was not effective in completely blocking thehybridization of a sequence or sequences shared by human chromosome 21and the centromeric regions of the other human acrocentric chromosomes.When a clone or clones containing such a sequence or sequences is or areeventually isolated, unlabeled DNA produced therefrom could be added tothe genomic blocking DNA to improve the specificity of the staining.

3c. Removal of Sequences.

3c.i. Hydroxyapatite. Single- and double-stranded nudeic acids havedifferent binding characteristics to hydroxyapatite. Suchcharacteristics provide a basis commonly used for fractionating nucleicacids. Hydroxyapatite is commerically available (eg. Bio-RadLaboratories, Richmond, Calif.). The fraction of genomic DNA containingsequences with a particular degree of repetition, from the highestcopy-number to single-copy, can be obtained by denaturing genomic DNA,allowing it to reassociate under appropriate conditions to a particularvalue of C_(o)t, followed by separation using hydroxyapatite. Thesingle- and double-stranded nudeic acid can also be discriminated by useof S1 nuclease. Such techniques and the concept of C_(o)t are explainedin Britten et al., “Analysis of Repeating DNA Sequences byReassociation”, in Methods in Enzymology, Vol. 29, pgs. 363418 (1974),which article is herein incorporated by reference.

The single-stranded nucleic acid fraction produced in 3a. or 3b. abovecan be separated by hydroxyapatite and used as a probe. Thus, thesequences that have been blocked (that become double-stranded) arephysically removed. The probe can then be stored until needed. The probecan then be used without additional blocking nucleic acid, or itsstaining contrast can perhaps be improved by additonal blocking.

3c.ii. Reaction with immobilized nucleic acid. Removal of particularsequences can also be accomplished by attaching single-stranded“absorbing” nucleic acid sequences to a solid support. Single-strandedsource nucleic acid is hybridized to the immobilized nucleic add. Afterthe hybridization, the unbound sequences are collected and used as theprobe. For example, human genomic DNA can be used to absorb repetitivesequences from human probes. One such method is described by Brison etal., “General Method for Cloning Amplified DNA by Differential Screeningwith Genomic Probes,” Molecular and Cellular Biology, Vol. 2, pgs.578-587 (1982). Accordingly, that reference is incorporated byreference. Briefly, minimally sheared human genomic DNA is bound todiazonium cellulose or a like support. The source DNA, appropriately cutinto fragments, is hybridized against the immobilized DNA to C_(o)tvalues in the range of about 1 to 100. The preferred stringency of thehybridization conditions may vary depending on the base composition ofthe DNA. Such a procedure could remove repetitive sequences fromchromosome-specific libraries, for example, those of Table I, to producea probe capable of staining a whole human chromosome.

3d. Blocking non-targeted sequences in the targeted genome. Blocking ofnon-targeted binding sites in the targeted genome by hybridization withunlabeled complementary sequences will prevent binding of labeledsequences in the probe that have the potential to bind to those sites.For example, hybridization with unlabeled genomic DNA will render thehigh-copy repetitive sequences in the target genome double-stranded.Labeled copies of such sequences in the probe will not be able to bindwhen the probe is subsequently applied.

In practice, several mechanisms combine to produce the stainingcontrast. For example, when blocking DNA is added to the probe as in 3babove, that which remains single-stranded when the probe is applied tothe target can bind to and block the target sequences. If the incubationof the probe with the blocking DNA is minimal, then the genomic DNAsimultaneously blocks the probe and competes with the probe for bindingsites in the target.

3e. Concept of Q. As mentioned in section 3b.i above, it is necessary toadd the correct amount of genomic DNA to achieve the best compromisebetween inhibiting the hybridization capacity of high-copy repeats inthe probe and reducing the desired signal intensity by inhibition of thebinding of the target-specific sequences. The following discussionpertains to use of genomic blocking DNA with probes produced by cloningor otherwise replicating stretches of DNA from the target region of thegenome. Thus, the probe contains a representative sampling of thesingle-copy, chromosome-specific repetitive sequences, and sharedrepetitive sequences found in the target. Such a probe might range incomplexity from 100 kb of sequence derived from a small region of thegenome, for example several closely spaced cosmid clones; to manymillions of bases, for example a combination of multiple libraries fromTable I. The discussion below is illustrative and can be extended toother situations where different blocking nucleic acids are used. Thefollowing discussion of Q is designed only to give general guidelines asto how to proceed.

The addition of unlabeled genomic DNA to a hybridization mix containinglabeled probe sequences increases the concentration of all of thesequences, but increases the concentration of the shared sequences by alarger factor than the concentration of the target-specific sequencesbecause the shared sequences are found elsewhere in the genome, whereasthe target-specific sequences are not. Thus, the reassociation of theshared sequences is preferentially enhanced so that the hybridization ofthe labeled copies of the shared sequences to the target ispreferentially inhibited.

To quantity this concept, first consider one of the sequences, repeat orsingle-copy, that hybridize specifically to the ith chromosome in ahybridization mixture containing a mass m_(p) of probe DNA from the ithchromosome library of Table 1 (for example) and m_(b) of unlabeledgenomic DNA. The number of labeled copies of the sequence isproportional to m_(p). However, the number of unlabeled copies isproportional to f_(i)m_(b), where f_(i) is the fraction of genomic DNAcontained on the ith chromosome. Thus, the ratio of unlabeled to labeledcopies of each of the sequences specific for the target chromosome, isf_(i)m_(b)/m_(p), which is defined herein as Q. For normal humanchromosomes, 0.016≦f_(i)≦0.08 [Mendelsohn et al., Science, 179:1126(1973)]. For representative examples described in Section VI.B (infra),f₄=0.066 and f₂₁=0.016. For a probe targeted at a region comprised of Lbase pairs, f_(i)=L/G where G is the number of base pairs in a genome(approximately 3×10⁹ bases for humans and other mammals). Thus, Q=(L/G)(m_(b)/m_(p)).

Now consider a shared sequence that is distributed more-or-lessuniformly over the genome, for example, Alu. The number of labeledcopies is proportional to m_(p), whereas the number of unlabeled copiesis proportional to m_(b). Thus, the ratio of unlabeled to labeled copiesis m_(b)/m_(p)=Q/f_(i). This is true for all uniformly distributedsequences, regardless of copy number. Thus adding genomic DNA increasesthe concentration of each specific sequence by the factor 1+Q, whereaseach uniformly distributed sequence is increased by the larger factor1+Q/f_(i). Thus, the reassociation rates of the shared sequences areincreased by a larger factor than those of the specific sequences by theaddition of genomic DNA.

It can be shown that roughly half of the beneficial effect of genomicDNA on relative reassociation rates is achieved when Q=1, and, by Q=5,there is essentially no more benefit to be gained by further increases.Thus, the protocol I hybridizations of Section VI.B infra keep Q≦5.

To illustrate the use of genomic blocking DNA, it is convenient toconsider a model of a genome wherein 50% of the DNA is comprised ofspecific sequences (both repetitive and single-copy) and the other 50%of the DNA is comprised of shared repetitive sequences that aredistributed uniformly over the genome. Thus, according to the model, ifthe target is L bases (that is, the probe contains fragmentsrepresenting L bases of the target area or areas of the genome),sequences containing L/2 bases will be specific to the target, and L/2will be shared with the entire genome.

Case I. The complexity of the probe is about 50 kb to about 100 kb. (Inthis case the complexity may be approximately equal to L since theprobability is that no repetitive sequences will typically occur withmore than a few copies in such a number of bases). Using a standardhybridization mixture (as exemplified in Section VI.B, infra), thetarget can be hybridized with about 2 ng of labeled probe DNA in 10 ulof hybridization mix, corresponding to approximately 1 pg/ul per kb ofspecific sequences (as used in Section VI.B, infra). Suppose thehybridization is to a slide containing 10⁴ cells (a typical number), andeach cell has about 6 pg of DNA, (typical for mammals). Then in thismodel calculation, there is 3 pg of shared repetitive sequences percell. Thus, for 10⁴ cells there are 3×10⁴ pg or 30 ng of sharedsequences on the slide. Similarly, there is 10⁴×0.5×10⁵×6/3×10⁹ pg=1 pgof target for the specific sequences. The probe contains 1/2×2 ng or 1ng of shared sequences and 1 ng of specific sequences. Therefore, thereis not enough probe to saturate the shared sequences in the target DNA,but enough to saturate the specific sequences. The signal from theshared sequences is spread at low intensity over the entire genomewhereas the specific signal is concentrated in a compact region. Thus,good contrast can be obtained without adding any blocking genomic DNA atall.

A great deal of genomic DNA can be added to improve the contrast withoutinterfering with the hybridization of the specific sequences, that is, Qremains low even if a great deal of genomic DNA is added.

Q=10⁵/3×10⁹ m _(b) /m _(p)=3×10⁻⁵ m _(b)/m_(p).

If a large amount of blocking nucleic acid, for example, 10 ug were used(according to the standard hybridization protocols exemplified inSection VI.B infra wherein the practical limit of total nudeic acid ison the order of 10 ug in a 10′ ul hybridization mixture) with the 2 ngof probe, then Q=3×10^(−5×10) ⁴ ng/2 ng=3/2×10⁻¹=0.15. Thus, Q is <1,and is so low that the blocking DNA cannot substantially interfere withthe desired signal. Increasing the amount of labeled probe nudeic acidto speed the hybridization would further decrease Q. In practice, onewould typically use 1 ug of blocking DNA for such a hybridization.

Case II. As the size of the target region is increased, the complexityof the probe necessarily is increased, and the amount of DNA in thehybridization mix needs to be increased in order to have a sufficientconcentration of each portion of specific sequence to hybridize. Also,if one desires to decrease the hybridization time of the procedure, theprobe concentration must be increased. In these situations, the increasein probe concentration results in an increase in the amount of sharedsequences in the hybridization mixture, which in turn increases theamount of hybridization that will occur to the shared sequences in thetarget area or areas, thereby reducing the contrast ratio.

With very high complexity probes spanning several entire chromosomes,L/G can approach 1. In order to stain such a portion of the genomewithin a reasonable time, for example, overnight, the concentration oflabeled nucleic acid needs to be increased, for example, 200 ng in 10 ulof hybridization mixture. Up to about 3000 ng of blocking DNA can beused and still keep Q≦5 [wherein the calculation is Q=5 =0.3 m_(b)/200ng or m_(b)=1000 ng/0.3=3,333 ng]. In practice, staining 25% and more ofthe human genome (for example, human chromosomes 1, 3 and 4) can beaccomplished with the blocking protocols described below, but thecontrast is less than for that achieved with probes for smaller regions.

III. Labeling the Nucleic Acid Fragments of the Heterogeneous Mixture

Several techniques are available for labeling single- anddouble-stranded nucleic acid fragments of the heterogeneous mixture.They include incorporation of radioactive labels, e.g. Harper et al.Chromosoma, Vol 83, pgs. 431-439 (1984); direct attachment offluorochromes or enzymes, e.g. Smith et al., Nucleic Acids Research,Vol. 13, pgs. 2399-2412 (1985), and Connolly et al., Nucleic AcidsResearch Vol. 13, pgs. 4485-4502 (1985); and various chemicalmodifications of the nucleic acid fragments that render them detectableimmunochemically or by other affinity reactions, e.g. Tchen et al.,“Chemically Modified Nucleic Acids as Immunodetectable Probes inHybridization Experiments,” Proc. Natl. Acad. Sci., Vol 81, pgs.3466-3470 (1984); Richardson et al., “Biotin and Fluorescent Labeling ofRNA Using T4 RNA Ligase,” Nucleic Acids Research, Vol. 11, pgs.6167-6184 (1983); Langer et al., “Enzymatic Synthesis of Biotin-LabeledPolynucleotides: Novel Nucleic Acid Affinity Probes,” Proc. Natl. Acad.Sci., Vol. 78, pgs. 6633-6637 (1981); Brigati et al., “Detection ofViral Genomes in Cultured Cells and Paraffin-Embedded Tissue SectionsUsing Biotin-Labeled Hybridization Probes,” Virology, Vol. 126, pgs.32-50 (1983); Broker et al., “Electron Microscopic Visualization of tRNAGenes with Ferritin-Avidin: Biotin Labels,” Nucleic Acids Research, Vol.5, pgs. 363-384 (1978); Bayer et al., “The Use of the Avidin BiotinComplex as a Tool in Molecular Biology,” Methods of Biochemical AnalysisVol. 26, pgs. 1-45 (1980) Kuhlmann, Immunoenzyme Techniques inCytochemistry (Weinheim, Basel, 1984). Langer-Safer et al., PNAS USA,79: 4381 (1982): Landegent et al., Exp. Cell Res., 153: 61 (1984); andHopman et al., Exp. Cell Res., 169: 357 (1987).

Exemplary labeling means include those wherein the probe fragments arebiotinylated, modified with N-acetoxy-N-2-acetylaminofluorene, modifiedwith fluorescein isothiocyanate, modified with mercury/TNP ligand,sulfonated, digoxigenenated or contain T-T dimers.

The key feature of “probe labeling” is that the probe bound to thetarget be detectable. In some cases, an intrinsic feature of the probenucleic acid, rather than an added feature, can be exploited for thispurpose. For example, antibodies that specifically recognize RNA/DNAduplexes have been demonstrated to have the ability to recognize probesmade from RNA that are bound to DNA targets [Rudkin and Stollar, Nature,265:472-473 (1977)]. The RNA used for such probes is unmodified. Probenucleic acid fragments can be extended by adding “tails” of modifiednucleotides or particular normal nucleotides. When a normal nudeotidetail is used, a second hybridization with nucleic acid complementary tothe tail and containing fluorochromes, enzymes, radioactivity, modifiedbases, among other labeling means, allows detection of the bound probe.Such a system is commerically available from Enzo Biochem (BiobridgeLabeling System; Enzo Biochem Inc., New York, N.Y.).

Another example of a means to visualize the bound probe wherein thenudeic acid sequences in the probe do not directly carry some modifiedconstituent is the use of antibodies to thymidine dimers. Nakane et al.,20 (2):229 (1987), illustrate such a method wherein thymine-thyminedimerized DNA (T-T DNA) was used as a marker for in situ hybridization.The hybridized T-T DNA was detected immunohistochemically using rabbitanti-T-T DNA antibody.

All of the labeling techniques disclosed in the above references may bepreferred under particular circumstances. Accordingly, the above-citedreferences are incorporated by reference. Further, any labelingtechniques known to those in the art would be useful to label thestaining compositions of this invention. Several factors govern thechoice of labeling means, including the effect of the label on the rateof hybridization and binding of the nucleic acid fragments to thechromosomal DNA, the accessibility of the bound probe to labelingmoieties applied after initial hybridization, the mutual compatibilityof the labeling moieties, the nature and intensity of the signalgenerated by the label, the expense and ease in which the label isapplied, and the like.

Several different high complexity probes, each labeled by a differentmethod, can be used simultaneously. The binding of different probes canthereby be distinguished, for example, by different colors.

IV. In Situ Hybridization

Application of the heterogeneous mixture of the invention to chromosomesis accomplished by standard in situ hybridization techniques. Severalexcellent guides to the technique are available, e.g., Gall and Pardue,“Nucleic Acid Hybridization in Cytological Preparations,” Methods inEnzymology. Vol. 21, pgs. 470-480 (1981); Henderson, “CytologicalHybridization to Mammalian Chromosomes,” International Review ofCytology, Vol. 76, pgs. 1-46 (1982); and Angerer, et al., “In SituHybridization to Cellular RNAs,” in Genetic Engineering: Principles andMethods Setlow and Hollaender, Eds., Vol. 7, pgs. 43-65 (Plenum Press,New York, 1985). Accordingly, these references are incorporated byreferences.

Three factors influence the staining sensitivity of the hybridizationprobes: (1) efficiency of hybridization (fraction of target DNA that canbe hybridized by probe), (2) detection efficiency (i.e., the amount ofvisible signal that can be obtained from a given amount of hybridizationprobe), and (3) level of noise produced by nonspecific binding of probeor components of the detection system.

Generally in situ hybridization comprises the following major steps: (1)fixation of tissue or biological structure to be examined, (2)prehybridization treatment of the biological structure to increaseaccessibility of target DNA, and to reduce nonspecific binding, (3)hybridization of the heterogeneous mixture of probe to the DNA in thebiological structure or tissue; (4) posthybridization washes to removeprobe not bound in specific hybrids, and (5) detection of the hybridizedprobes of the heterogeneous mixture. The reagents used in each of thesesteps and their conditions of use vary depending on the particularsituation.

The following comments are meant to serve as a guide for applying thegeneral steps listed above. Some experimentation may be required toestablish optimal staining conditions for particular applications.

In preparation for the hybridization, the probe, regardless of themethod of its production, may be broken into fragments of the sizeappropriate to obtain the best intensity and specificity ofhybridization. As a general guideline concerning the size of thefragments, one needs to recognize that if the fragments are too longthey are not able to penetrate into the target for binding and insteadform aggregates that contribute background noise to the hybridization;however, if the fragments are too short, the signal intensity isreduced.

Under the conditions of hybridization exemplified in Section VI.Bwherein human genomic DNA is used as an agent to block the hybridizationcapacity of the high copy shared repetitive sequences, the preferredsize range of the probe fragments is from about 200 bases to about 600.The preferred hybridization temperature is about 30° C. to about 45° C.,more preferably about 35° C. to about 40° C., and still more preferablyabout 37° C.; preferred washing temperature range is from about 40° C.to about 50° C., more preferably about 45° C.

The size of the probe fragments is checked before hybridization to thetarget; preferably the size of the fragments is monitored byelectrophoresis, more preferably by denaturing agarose gelelectrophoresis.

Fixatives include acid alcohol solutions, acid acetone solutions,Petrunkewitsch's reagent, and various aldehydes such as formaldehyde,paraformaldehyde, glutaraldehyde, or the like. Preferably,ethanol-acetic acid or methanol-acetic acid solutions in about 3:1proportions are used to fix the chromosomes in metaphase spreads. Forcells or chromosomes in suspension, a fixation procedure disclosed byTrask, et al., in Science, Vol. 230, pgs. 1401-1402 (1985), is useful.Accordingly, Trask et al., is incorporated by reference. Briefly, K₂CO₃and dimethylsuberimidate (DMS) are added (from a 5×concentrated stocksolution, mixed immediately before use) to a suspension containing about5×10⁶ nuclei/ml. Final K₂CO₃ and DMS concentrations are 20 mM and 3 mM,respectively. After 15 minutes at 25° C., the pH is adjusted from 10.0to 8.0 by the addition of 50 microliters of 100 mM citric acid permilliliter of suspension. Nuclei are washed once by centrifugation (300g, 10 minutes, 4° C. in 50 mM kCl, 5 mM Hepes buffer, at pH 9.0, and 10mM MgSO₄).

A preferred fixation procedure for cells or nuclei in suspension isdisclosed by Trask et al., Hum. Genet. 78:251-259 (1988), which articleis herein incorporated by reference. Briefly, nuclei are fixed for about10 minutes at room temperature in 1% paraformaldehyde in PBS, 50′ mMMgSO₄, pH 7.6 and washed twice. Nuclei are resuspended in isolationbuffer (IB) (50 mM KCl, 5 mM HEPES, 10 mM MgSO₄, 3 mM dithioerythritol,0.15 mg/ml RNase, pH 8.0)/0.05% Triton X-100 at 10⁸/ml.

Frequently before in situ hybridization chromosomes are treated withagents to remove proteins. Such agents include enzymes or mild acids.Pronase, pepsin or proteinase K are frequently used enzymes. Arepresent- ative acid treatment is 0.02-0.2 N HCl, followed by hightemperature (e.g., 70° C.) washes. Optimization of deproteinizationrequires a combination of protease concentration and digestion time thatmaximizes hybridization, but does not cause unacceptable loss ofmorphological detail. Optimum conditions vary according to tissue typesand method of fixation. Additional fixation after protease treatment maybe useful. Thus, for particular applications, some experimentation maybe required to optimize protease treatment.

In some cases pretreatment with RNase may be desirable to removeresidual RNA from the target. Such removal can be accomplished byincubation of the fixed chromosomes in 50-100 microgram/milliliter RNasein 2×SSC (where SSC is a solution of 0.15M NaCL and 0.015M sodiumcitrate) for a period of 1-2 hours at room temperature.

The step of hybridizing the probes of the heterogeneous probe mixture tothe chromosomal DNA involves (1) denaturing the target DNA so thatprobes can gain access to complementary single-stranded regions, and (2)applying the heterogeneous mixture under conditions which allow theprobes to anneal to complementary sites in the target. Methods fordenaturation include incubation in the presence of high pH, low pH, hightemperature, or organic solvents such as formamide, tetraalkylammoniumhalides, or the like, at various combinations of concentration andtemperature. Single-stranded DNA in the target can also be produced withenzymes, such as, Exonuclease III [van Dekken et al., Chromosoma (Berl)97:1-5 (1988)]. The preferred denaturing procedure is incubation forbetween about 1-10 minutes in formamide at a concentration between about35-95 percent in 2×SSC and at a temperature between about 25-70° C.Determination of the optimal incubation time, concentration, andtemperature within these ranges depends on several variables, includingthe method of fixation and type of probe nucleic acid (for example, DNAor RNA).

After the chromosomal DNA is denatured, the denaturing agents aretypically removed before application of the heterogeneous probe mixture.Where formamide and heat are the primary denaturing agents, removal isconveniently accomplished by several washes with a solvent, whichsolvent is frequently chilled, such as a 70%, 85%, 100% cold ethanolseries. Alternatively the composition of the denaturant can be adjustedas appropriate for the in situ hybridization by addition of otherconstituents or washes in appropriate solutions. The probe and targetnucleic acid may be denatured simultaneously by applying thehybridization mixture and then heating to the appropriate temperature.

The ambient physiochemical conditions of the chromosomal DNA and probeduring the time the heterogeneous mixture is applied is referred toherein as the hybridization conditions, or annealing conditions. Optimalhybridization conditions for particular applications can be adjusted bycontrolling several factors, including concentration of theconstituents, incubation time of chromosomes in the heterogeneousmixture, and the concentrations, complexities, and lengths of thenucleic acid fragments making up the heterogeneous mixture. Roughly, thehybridization conditions must be sufficiently close to the meltingtemperature to minimize nonspecific binding. On the other hand, theconditions cannot be so stringent as to reduce correct hybridizations ofcomplementary sequences below detectable levels or to requireexcessively long incubation times.

The concentrations of nucleic acid in the hybridization mixture is animportant variable. The concentrations must be high enough so thatsufficient hybridization of respective chromosomal binding sites occursin a reasonable time (e.g., within hours to several days). Higherconcentrations than that necessary to achieve adequate signals should beavoided so that nonspecific binding is minimized. An important practicalconstraint on the concentration of nucleic acid in the probe in theheterogeneous mixture is solubility. Upper bounds exist with respect tothe fragment concentration, i.e., unit length of nudeic acid per unitvolume, that can be maintained in solution and hybridize effectively.

In the representational examples described in Section VI.B (infra), thetotal DNA concentration in the hybridization mixture had an upper limiton the order of 1 ug/ul. Probe concentrations in the range of 1-20 ng/ulwere used for such whole chromosome staining. The amount of genomicblocking DNA was adjusted such that Q was less than 5. At the low end ofprobe concentration, adequate signals were obtained with a one hourincubation, that is, a time period wherein the probe and blocking DNAare maintained together before application to the targeted material, toblock the high-copy sequences and a 16 hour hybridization. Signals werevisible after two hours of hybridization. The best results (brightsignals with highest contrast) occurred after a 100 hour hybridization,which gave the low-copy target-specific sequences more opportunity tofind binding sites. At the high end of the probe concentration, brightsignals are obtained after hybridizations of 16 hours or less; thecontrast was reduced since more labeled repetitive sequences wereincluded in the probe.

The fixed target object can be treated in several ways either during orafter the hybridization step to reduce nonspecific binding of probe DNA.Such treatments include adding nonprobe, or “carrier”, DNA to theheterogeneous mixture, using coating solutions, such as Denhardt'ssolution (Biochem. Biophys. Res. Commun., Vol. 23, pgs. 641-645 (1966),with the heterogeneous mixture, incubating for several minutes, e.g.,5-20, in denaturing solvents at a temperature 5-10° C. above thehybridization temperature, and in the case of RNA probes, mild treatmentwith single strand RNase (e.g., 5-10 micrograms per millileter RNase) in2×SSC at room temperature for 1 hour).

V. Chromosome-Specific Staining Reagents Comprising Selected Single-CopySequences

V.A. Making and Using a Staining Reagent Specific to Human Chromosome 21

V.A.1. Isolation of Chromosome 21 and Construction of a Chromosome21-Specific Library

DNA fragments from human chromosome-specific libraries are availablefrom the National Laboratory Gene Library Project through the AmericanType Culture Collection (ATCC), Rockville, Md. DNA fragments fromchromosome 21 were generated by the procedure described by Fuscoe etal., in “Construction of Fifteen Human Chromosome-Specific DNA Librariesfrom Flow-Purified Chromosomes,” Cytogenet. Cell Genet., Vol. 43, pgs.79-86 (1986), which reference is incorporated by reference. Briefly, ahuman diploid fibroblast culture was established from newborn foreskintissue. Chromosomes of the cells were isolated by the MgSO₄ method ofvan den Engh et al., Cytometery, Vol. 5, pgs. 108-123 (1984), andstained with the fluorescent dyes—Hoechst 33258 and Chromomycin A3.Chromosome 21 was purified on the Lawrence Livermore National Laboratoryhigh speed sorter, described by Peters et al., Cytometry, Vol. 6, pgs.290-301 (1985).

After sorting, chromosome concentrations were approximately 4×10⁵/ml.Therefore, prior to DNA extraction, the chromosomes (0.2-1.0×10⁶) wereconcentrated by centrifugation at 40,000×g for 30 minutes at 4° C. Thepellet was then resuspended in 100 microliters of DNA isolation buffer(15 mM NaCl, 10 mM EDTA, 10 mM Tris HCl pH 8.0) containing 0.5% SDS and100 micrograms/ml proteinase K. After overnight incubation at 37° C.,the proteins were extracted twice with phenol:chloroform: isoamylalcohol (25:24:1) and once with chloroform:isoamyl alcohol (24:1).Because of the small amounts of DNA, each organic phase was reextractedwith a small amount of 10 mM Tris pH 8.0, 1 mM EDTA (TE). Aqueous layerswere combined and transferred to a Schleicher and Schuell mini-collodionmembrane (#UHO20/25) and dialyzed at room temperature against TE for 6-8hours. The purified DNA solution was then digested with 50 units ofHindIII (Bethesda Research Laboratories, Inc.) in 50 mM NaCl, 10 mM TrisHCl pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol. After 4 hours at 37°, thereaction was stopped by extractions with phenol and chloroform asdescribed above. The aqueous phase was dialyzed against water overnightat 4° C. in a mini-collodion bag and then 2 micrograms of Charon 21Aarms cleaved with HindIII and treated with calf alkaline phosphatase(Boehringer Mannheim) were added. This solution was concentrated undervacuum to a volume of 50-100 microliters and transferred to a 0.5 mlmicrofuge tube where the DNA was precipitated with one-tenth volume 3Msodium acetate pH 5.0 and 2 volumes ethanol. The precipitate wascollected by centrifugation, washed with cold 70% ethanol, and dissolvedin 10 microliters of TE.

After allowing several hours for the DNA to dissolve, 1 microliter of10×X ligase buffer (0.5M Tris HCl pH 7.4, 0.1 M MgCl₂, 0.1Mdithiothreitol, 10 mM ATP, 1 mg/ml bovine serum albumin) and 1 unit ofT4 ligase (Bethesda Research Laboratory, Inc.) were added. The ligationreaction was incubated at 10° C. for 16-20 hours and 3 microliteraliquots were packaged into phage particles using in vitro extractsprepared from E. coli strains BHB 2688 and BHB 2690, described by Hohnin Methods in Enzymology, Vol. 68, pgs. 299-309 (1979) MolecularCloning: A Laboratory Manual, (Cold Spring Harbor Laboratory, New York,1982). Briefly, both extracts were prepared by sonication and combinedat the time of in vivo packaging. These extracts packaged wild-typelambda DNA at an efficiency of 1-5×10⁸ plaque forming units (pfu) permicrogram. The resultant phage were amplified on E. coli LE392 at adensity of approximately 10⁴ pfu/150 mm dish for 8 hours to preventplaques from growing together and to minimize differences in growthrates of different recombinants. The phage were eluted from the agar in10 ml SM buffer (50 mM Tris HCl pH 7.5, 10 mM MgSO₄, 100 mM NaCl, 0.01%gelatin) per plate by gentle shaking at 4° C. for 12 hours. The plateswere then rinsed with an additional 4 ml of SM. After pelleting cellulardebris, the phage suspension was stored over chloroform at 4° C.

V.A.2. Construction and Use of Chromosome 21-Specific Stain for StainingChromosome 21 of Human Lymphocytes

Clones having unique sequence inserts are isolated by the method ofBenton and Davis, Science, Vol. 196, pgs. 180-182, (1977). Briefly,about 1000 recombinant phage are isolated at random from the chromosome21-specific library. These are transferred to nitrocellulose and probedwith nick translated total genomic human DNA.

Of the clones which do not show strong hybridization, approximately 300are picked which contain apparent unique sequence DNA. After theselected clones are amplified, the chromosome 21 insert in each done is³²P labeled and hybridized to Southern blots of human genomic DNAdigested with the same enzyme used to construct the chromosome 21library, i.e., Hind III. Unique sequence containing clones arerecognized as those that produce a single band during Southern analysis.Roughly, 100 such clones are selected for the heterogeneous mixture. Theunique sequence clones are amplified, the inserts are removed by HindIII digestions, and the inserts are separated from the phage arms by gelelectrophoresis. The probe DNA fragments (i.e., the unique sequenceinserts) are removed from the gel and biotinylated by nick translation(e.g., by a kit available from Bethesda Research Laboratories). LabeledDNA fragments are separated from the nick translation reaction usingsmall spin columns made in 0.5 ml Eppendorph tubes filled with SephadexG-50 (medium) swollen in 50 mM Tris, 1 mM EDTA, 0.1% SDS, at pH 7.5.Human lymphocyte chromosomes are prepared following Harper et al, Proc.Natl. Acad. Sci. Vol. 78, pgs. 4458-4460 (1981). Metaphase andinterphase cells were washed 3 times in phosphate buffered saline, fixedin methanol-acetic acid (3:1) and dropped onto cleaned microscopeslides. Slides are stored in a nitrogen atmosphere at −20° C.

Slides carrying interphase cells and/or metaphase spreads are removedfrom the nitrogen, heated to 65° C. for 4 hours in air, treated withRNase (100 micrograms/ml for 1 hour at 37° C.), and dehydrated in anethanol series. They are then treated with proteinase K (60 ng/ml at 37°C. for 7.5 minutes) and dehydrated. The proteinase K concentration isadjusted depending on the cell type and enzyme lot so that almost nophase microscopic image of the chro- mosomes remains on the dry slide.The hybridization mix consists of (final concentrations) 50 percentformamide, 2×SSC, 10 percent dextran sulfate, 500 micrograms/ml carrierDNA (sonicated herring sperm DNA), and 2.0 microgram/ml biotin-labeledchromosome 21-specific DNA. This mixture is applied to the slides at adensity of 3 microliters/cm² under a glass coverslip and sealed withrubber cement. After overnight incubation at 37° C., the slides arewashed at 45° C. (50% formamide-2×SSC pH 7, 3 times 3 minutes; followedby 2×SSC pH 7, 5 times 2 minutes) and immersed in BN buffer (0.1 M Nabicarbonate, 0.05 percent NP-40, pH 8). The slides are never allowed todry after this point.

The slides are removed from the BN buffer and blocked for 5 minutes atroom temperature with BN buffer containing 5% non-fat dry milk(Carnation) and 0.02% Na Azide (5 microliter/cm² under plasticcoverslips). The coverslips are removed, and excess liquid brieflydrained and fluorescein-avidin DCS (3 microgram/ml in BN buffer with 5%milk and 0.02% NaAzide) is applied (5 microliter/cm²). The samecoverslips are replaced and the slides incubated 20 minutes at 37° C.The slides are then washed 3 times for 2 minutes each in BN buffer at45° C. The intensity of biotin-linked fluorescence is amplified byadding a layer of biotinylated goat anti-avidin antibody (5 microgram/mlin BN buffer with 5% goat serum and 0.02% Na Azide), followed, afterwashing as above, by another layer of fluorescein-avidin DCS.Fluorescein-avidin DCS, goat antiavidin and goat serum are all availablecommercially, e.g., Vector Laboratories (Burlingame, Calif.). Afterwashing in BN, a fluorescence antifade solution, p-phenylenediamine (1.5microliter/cm² of coverslip) is added before observation. It isimportant to keep this layer thin for optimum microscopic imaging. Thisantifade significantly reduced fluorescein fading and allows continuousmicroscopic observation for up to 5 minutes. The DNA counterstains (DAPIor propidium iodide) are included in the antifade at 0.25-0.5microgram/ml.

The red-fluorescing DNA-specific dye propidium iodide (PI) is used toallow simultaneous observation of hybridized probe and total DNA. Thefluorescein and PI are excited at 450-490 nm (Zeiss filter combination487709). Increasing the excitation wavelength to 546 nm (Zeiss filtercombination 487715) allows observation of the PI only. DAPI, a bluefluorescent DNA-specific stain excited in the ultraviolet (Zeiss filtercombination 487701), is used as the counterstain when biotin-labeled andtotal DNA are observed separately. Metaphase chromosome 21s are detectedby randomly located spots of yellow distributed over the body of thechromosome.

V.B. Improved Method for Efficiently Selecting Chromosome 21 Single-CopySequences

Fuscoe et al., Genomics, 5:100-109 (1989) provides more efficientprocedures than the method described immediately above (V.A.2) forselecting large numbers of single-copy sequence or very low copy numberrepeat sequence clones from recombinant phage libraries and demonstratestheir use to stain chromosome 21. Said article is hereby incorporated byreference. Briefly, clones were selected from the Charon 21A libraryLL21NS02 (made from DNA from human chromosome 21) using two basicprocedures. In the first, the phage library was screened in two stagesusing methods designed to be more sensitive to the presence ofrepetitive sequences in the clones than the method of Section V.A.2. Theselected clones were then subcloned into plasmids. The 450 inserts thusselected form the library pBS-U21. The second was in a multistep processin which: 1) Inserts from LL21NS02 were subcloned into Bluescribeplasmids, 2) plasmids were grown at high density in bacterial colonieson nitrocellulose filters and 3) radioactive human genomic DNA washybridized to the plasmid DNA on nitrocellulose filters at lowstringency in two steps and 4) plasmids having inserts that failed tohybridize were selected as potentially carrying single-copy sequences.Fifteen hundred and thirty colonies were picked in this manner to formthe library pBS-U21/1530.

Southern analysis indicated that the second procedure was more effectiveat recognizing repetitive sequence than the first. Fluorescence in situhybridization with DNA from pBS-U21/1530 allowed specific, intensestaining of the number 21 chromosomes in metaphase spreads made fromhuman lymphocytes. Hybridization with pBS-U21 gives less specificstaining of chromosome 21. Details concerning the Fuscoe et al. methodof selecting single-copy sequence or very low repeat sequence probesfrom recombinant libraries can be found in found in Fuscoe et al., id.

V.C. Hybridization with a Collection of Chromosome 4 Single-CopySequences

Chromosome 4 Single-Copy Sequences. One hundred and twenty clonescarrying chromosome 4-specific single-copy sequence inserts selectedfrom the Charon 21A library LL04NS01 (ATCC accession number 57700; VanDilla et al., supra; see Table 1) were supplied by C. Gilliam (HarvardUniversity) [Gilliam et al., Nucleic Acids Res. 15:1445 (1987)]. Thehuman inserts were all about 3 kilobases (kb) in length, so the ratio ofinsert to vector DNA was <0.1. Total phage DNA was produced from eachclone individually using DEAE-cellulose columns (Whatman DE-52) [Helmset al., DNA 4:39 (1985)]. DNA pooled from the 120 clones wasbiotinylated by nick-translation with biotin-11-dUTP (Bethesda ResearchLaboratories) and recovered at a concentration of about 20 nanograms permicroliter (ng/ul) using Sephadex G-50 spin columns.

Cells. Metaphase spreads from human lymphocytes were prepared frommethotrexate-synchronized cultures by using the procedure of Harper etal., supra. The cells were fixed in methanol/acetic acid, 3:1. Slideswere stored at −20° C. in plastic bags filled with nitrogen gas.

In Situ Hybridization: Single-Copy Hybridization. Hybridization wasaccomplished by using a modification of the procedure described byPinkel et al., PNAS USA, 83: 2934 (1986). The slide mounted cells weretreated with RNase [100 micrograms per milliliter (ug/ml) in 0.3 molar(M) sodium chloride (NaCl)/30 millimolar (mM) sodium citrate at 37° C.for 1 hr), dehydrated in a 70%/85%/100% ethanol series, treated withproteinase K (0.3-0.6 ug/ml in 20 mM Tris/2 mM CaCl₂, pH 7.5, for 7.5min at 37° C.), and fixed [4% paraformaldehyde in phosphate-bufferedsaline (PBS; in g/liter, KCl, 0.2; KH₂PO₄, 0.2; NaCl, 8; Na₂HPO₄.7H₂O,2.16) plus 50′ mM MgCl₂ for 10 min at room temperature]. The DNA in thetarget cells was denatured by immersion in 70% formamide/2×SSC (0.3 MNaCl/30 mM sodium citrate) at pH 7, for 2 min at 70° C. Thehybridization mixture [10 ul total volume consisting of 50% formamide,0.3 M NaCl/30 mM sodium citrate (final concentration), 10% dextransulfate, 50 ug of sonicated herring DNA per ml, and 3-6 ng ofbiotinylated chromosome 4 unique sequences (40-80 ng of total phageDNA)] was then denatured (70° C. for 5 min) and applied. Hybridizationwas at 37° C. overnight (16 hr). Slides were washed in three changes of50% formamide/0.3 M NaCl/30 mM sodium citrate (final concentration), pH7, at 45° C. for 5 min each and once in PN buffer (a mixture of 0.1 MNaH₂PO₄ and 0.1 M Na₂HPO4 to give pH 8/0.1% Nonidet P-40). The slideswere then treated with alternating layers of fluoresceinated avidin andbiotinylated goat antiavidin, both at 5 ug/ml in PNM buffer (PNbuffer/5% non-fat dry milk/0.02% sodium azide, centrifuged to removesolids), for 20 min each at room temperature until three layers ofavidin were applied. The avidin and goat anti-avidin treatments wereseparated by three washes of 3 min each in PN buffer [avidin (DCS grade)and anti-avidin from Vector Laboratories (Burlingame, Calif.)]. Afterthe final avidin treatment, a fluorescence antifade solution [Johnsonand Noqueria, J. Immunol. Methods, 43:349 (1981)] containing 1 ug of4′,6-amidino-2-phenylindole or propidium iodide per ml was applied as acounterstain (1.5 ul/cm² under a no. 1 coverslip).

Results. As shown in FIG. 4H, individual hybridization sites could belocated to within a fraction of the width of a chromatid after overnighthybridization (16 hr) and application of three layers of avidin.Analysis of three spreads from the hybridization with the 120 uniquesequence probes at a total probe concentration of 1.5 pg/ul per kilobaseof human insert, showed 222 fluorescent spots out of the 1440 possibleon the number 4 chromosomes (120 target sites per chromatid×4 chromatidsper metaphase×3 metaphases). Thus, the hybridization efficiency was 15%.There were 814 total spots on all of the chromosomes giving ahybridization specificity of 27%. The experiment demonstrates thatsubstantial hybridization can occur with single copy probes at low probeconcentrations in overnight hybridizations. The contrast ratio ofchromosome 4 relative to the rest of the${{chromosomes}\quad {was}\quad {thus}\text{:}\frac{{{spots}/{length}}\quad {of}\quad {chromosome}\quad 4}{{{spots}/{length}}\quad {of}\quad {all}\quad {chromosomes}}} = {\frac{222/{.06}}{814/1.0} = {{approximately}\quad 4.}}$

(Chromosome 4 comprises about 6% of the genome.)

VI. Incapacitating Shared Repetitive Sequences

VI.A. Chromosome 21-Specific Staining Using Blocking DNA

High concentrations of unlabeled human genomic DNA and lambda phage DNAwere used to inhibit the binding of repetitive and vector DNA sequencesto the target chromosomes. Heavy proteinase digestion and subsequentfixation of the target improved access of probes to target DNA.

Human metaphase spreads were prepared on microscope slides with standardtechniques and stored immediately in a nitrogen atmosphere at −20° C.

Slides were removed from the freezer and allowed to warm to roomtemperature in a nitrogen atmosphere before beginning the stainingprocedure. The warmed slides were first treated with 0.6 microgram/mlproteinase K in P buffer (20 mM Tris, 2 mM CaCl₂ at pH 7.5) for 7.5minutes, and washed once in P buffer. The amount of proteinase K usedneeds to be adjusted for different batches of slides. After denaturingthe slides were stored in 2×SSC. A hybridization mix was prepared whichconsisted of 50% formamide, 10% dextran sulfate, 1% Tween 20, 2×SSC, 0.5mg/ml human genomic DNA, 0.03 mg/ml lambda DNA, and 3 microgram/mlbiotin labeled probe DNA. The probe DNA consisted of the highest densityfraction of phage from the chromosome 21 Hind III fragment library (ATCCaccession number 57713), as determined by a cesium chloride gradient.(Both insert and phage DNA of the probe were labeled by nicktranslation.) The average insert size (amount of chromosome 21 DNA), asdetermined by gel electrophoresis was about 5 kilobases. No attempt wasmade to remove repetitive sequences from the inserts or to isolate theinserts from the lambda phage vector. The hybridization mix wasdenatured by heating to 70° C. for 5 minutes followed by incubation at37° C. for 1 hour. The incubation allows the human genomic DNA andunlabeled lambda DNA in the hybridization mix to block the humanrepetitive sequences and vector sequences in the probe.

The slide containing the human metaphase spread was removed from the2×SSC and blotted dry with lens paper. The hybridization mix wasimmediately applied to the slide, a glass cover slip was placed on theslide with rubber cement, and the slide was incubated overnight at 37°C. Afterwards preparation of the slides proceeded as described inSection V.B. (wherein chromosome 21 DNA was stained with fluorescein andtotal chromosomal DNA counterstained with DAPI). FIGS. 1A-C illustratethe results. FIG. 1A is a DAPI image of the human metaphase spreadobtained with a computerized image analysis system. It is a binary imageshowing everything above threshold as white, and the rest as black. Theprimary data was recorded as a gray level image with 256 intensitylevels. (Small arrows indicate the locations of the chromosome 21s.)FIG. 1B is a fluorescein image of the same spread as in FIG. 1A, againin binary form. (Again, small arrows indicate the locations of thechromosome 21s.) FIG. 1C illustrates the positions of the chromosome 21safter other less densely stained objects were removed by standard imageprocessing techniques.

VI.B. Detection of Trisomy 21 and Translocations of Chromosome 4 UsingBluescribe Plasmid Libraries

As illustrated in Section VI.A., a human chromosome-specific library,including its shared repetitive sequences, can be used to stain thatchromosome if the hybridization capacity of the shared repetitivesequences is reduced by incubation with unlabeled human genomic DNA. InSection VI.A., the nucleic acid sequences of the heterogeneous mixturewere cloned in the phage vector Charon 21A, in which the ratio of insertof vector DNA is about 0.1 (4 kb average insert to 40 kb of vector). Inthis section, we demonstrate that transferring the same inserts to asmaller cloning vector, the about 3 kb Bluescribe plasmid, whichincreases the ratio of insert to vector DNA to 0.5, improved thespecificity and intensity of the staining.

As previously discussed, incubation of the probe can be carried out withthe probe alone, with the probe mixed with unlabeled genomic DNA, andwith the probe mixed with unlabeled DNA enriched in all or some sharedrepetitive sequences. If unlabeled genomic DNA is added, then it isimportant to add enough to incapacitate sufficiently the sharedrepetitive sequences in the probe. However, the genomic DNA alsocontains unlabeled copies of the sequences, the hybridization of whichis desired. As explained above, Q is herein defined as the ratio ofunlabeled to labeled copies of the chromosome- specific sequences in thehybridization mixture.

Cells. Metaphase spreads from human lymphocytes were prepared frommethotrexate-synchronized cultures by using the procedure of Harper etal. supra. These and all other cells used in this example were fixed inmethanol/acetic add, 3:1. Other human lymphocyte cultures wereirradiated with ⁶⁰Co gamma rays and stimulated with phytohemagglutnin.Colcemid was added 48 hr after stimulation and metaphase spreads wereprepared 4 hr later. Metaphase spreads and interphase cells fromlymphoblastoid cells (GM03716A; Human Mutant Cell Repository, Camden,N.J.) carrying trisomy 21 were prepared after a 4-hr colcemid block.Interphase cells from the cell line RS4;11 carrying t(4;11) andisochromosome 7q were harvested, fixed in methanol/acetic add, anddropped onto slides [Strong et al., Blood, 65:21 (1985)]. Slides werestored at −20° C. in plastic bags filled with nitrogen gas.

pBS-4. The entire chromosome 4 library LL04NS02 (ATCC accession number57745; Van Dilla et al., supra) was subcloned into Bluescribe plasmids(Stratagene La Jolla, Calif.) to form the library pBS4. The averageinsert to vector DNA ratio in pBS-4 is about 1. The plasmid library wasamplified in bulk and the DNA was extracted using DEAE-cellulose columns(Whatman DE-52) [Helms et al., DNA, 4:39 (1985)]. The DNA was thenbiotinylated by nick translation with biotin-11-dUTP (Bethesda ResearchLaboratories) and recovered at a concentration of about 20 ng/ul usingSephadex G-50 spin columns. In some experiments, the biotinylated DNAwas concentrated by ethanol precipitation to achieve higher probeconcentrations.

pBS-21. The entire chromosome 21 library LL21NS02 (ATCC accession number57713; Van Dilla et al., supra) was subcloned into Bluescribe plasmidsto form the library pBS-21. This library was amplified and biotinylatedas described above for pBS4.

Human genomic DNA. Placental DNA (Sigma) was treated with proteinase K,extracted with phenol, and sonicated to a size range of 200-600 basepairs (bp).

Whole Library Hybridization. Hybridization was as above in section V.Cexcept that RNase, proteinase K, and paraformaldehyde were not used. Theamount of probe and genomic DNA in the hybridization mixture and thelength of the hybridization varied as described in Results. All probeconcentrations refer to the human insert DNA unless otherwise noted. DNAconcentrations were determined by fluorometric analysis (HoefferScientific Instruments, San Francisco). Incubation of the hybridizationmixture prior to hybridization followed two different protocols asindicated immediately below.

Protocol I. The hybridization mixture (10 ul) contained 10-150 ng ofbiotinylated human DNA (20-300 ng of total plasmid DNA) and 0-10 ug ofunlabeled genomic DNA. The mixture was heated to denature the DNA andincubated at 37° C. for a time t before it was added to the slide.Hybridization times ranged from 2 to 110 hr.

Protocol II. Protocol II was identical to Protocol I except that anadditional aliquot of freshly denatured genomic DNA was added to thehybridization mixture after an incubation time t. The mixture was thenincubated an additional time t prior to starting the hybridization. Thevolume of the hybridization mixture was increased <20% by the additionalgenomic DNA.

Microscopy. Quantitative fluorescence measurements were performed usinga video camera on the microscope and a digital image processing system,[Trask et al., Human Genet., 78:251 (1988)] Results. FIG. 4A showshybridization of pBS-4 to a human metaphase spread with a probeconcentration of 1 ng/ul. No genomic DNA was used and the hybridizationmixture was applied immediately after denaturation. All of thechromosomes are stained, except near many centromeres, with two copiesof chromosome 4 being stained most heavily. All the chromosomes arestained along most of their lengths due to sequences in the probe whichare shared with other chromosomes. Unstained regions, noted by arrows,show locations for which homologous sequences are not present in pBS-4.The unstained regions are mostly centromeric and along the long arm ofthe Y chromosome. Blocks of repetitive DNA specific to those sites areknown to exist.

The visible contrast on chromosome 4 is the result of the interaction ofseveral factors. (i) All of the DNA in chromosome 4 is potential targetfor sequences in the probe, whereas only those sequences on the otherchromosomes that are shared with chromosome 4 can bind probe. (ii) Thehybridization time and probe concentration were high enough to allowsignificant binding of the specific sequences in the probe. (iii) Theratio of probe to target sequences is higher for the specific sequencesthan for the shared sequences [Ten nanograms of chromosome 4 DNA washybridized to about 200 ng of human DNA target (4×10⁴ cells), 13 ng ofwhich is chromosome 4. Thus, the ratio of probe to target for thespecific sequences was about 1, whereas for the shared sequences it wasabout 0.05.]

The contrast can be increased by allowing the denatured probe DNA topartially reassociate prior to adding it to the slide, preferentiallydepleting the single-stranded high-copy (predominantly the shared)sequences in the probe [Cantor & Schimmel, Biophysical Chemistry: TheBehavior of Biological Macromolecules, (part III, p. 1228) (Freeman1980)]. A significant increase in staining specificity resulting fromprobe reassociation was observed experimentally for chromosome 4 using ahybridization mixture with 1 ng of probe per microliter (ul) and a 24 hrincubation at 37° C. prior to in situ hybridization (not shown).Likewise, hybridization after a 24 hr incubation of 4 ng of chromosome21 probe per ul resulted in a substantial contrast ratio. That resultindicates that at such concentrations the chromosome-specific sequencesremain substantially single stranded for times on the order of days inthe hybridization mixture. It also demonstrates that other mechanismsthat might inactivate the probe are not significant during theincubation.

FIGS. 4B and 4C show the result of a protocol I hybridization [0.8 ng ofprobe per ul and 24 ng of genomic DNA per ul (Q=2); 1-hr probeincubation and 110-hr hybridization]. Quantitative image analysis showsthat the intensity per unit length of the FITC fluorescein on chromosome4 is approximately 20 times that of the other chromosomes, that is thecontrast ratio is 20:1. Two layers of avidin-fluorescein isothiocyanatehave been used here to make the non-target chromosomes sufficientlybright to be measured accurately. However, the number 4 chromosomes canbe recognized easily after a single layer.

FIG. 4D demonstrates detection of a radiation-induced translocationinvolving chromosome 4 in human lymphocytes [protocol I, 1′ ng of probeper ul and 76 ng of genomic DNA per ul (Q=5); 1-hr probe incubation and16-hr hybridization]. The contrast ratio was about 5. The hybridizationintensity and specificity shown in FIG. 4D are such that even smallportions of the involved chromosome can be detected.

The ease with which translocations can be recognized offers theopportunity for translocation detection by automated means, such as,computerized microscopy or flow cytometry. [See Section VIII infra forelaboration concerning automated detection means.]

FIG. 4E shows that the normal and two derivative chromosomes resultingfrom the translocation between chromosomes 4 and 11 [t(4;11)] in cellline RS4;11 can be detected in interphase nuclei as three distinctdomains [protocol I, 13.5 ng of probe per ul and 800 ng of genomic DNAper ul (Q=5); 1-hr probe incubation and 16-hr hybridization]. Theincreased probe concentration resulted in brighter signals relative toFIG. 4D. Approximately half of the cells clearly show the presence ofthree nuclear domains, presumably produced by the two portions of theinvolved chromosome 4 and the intact normal chromosome. The domains inthe other nuclei may have been obscured by the nuclear orientation inthese two-dimensional views, by nuclear distortion that occurred duringslide preparation, or because the domains were too close to each otherto be distinguished. Hybridization using procedures that preservethree-dimensional morphology may resolve these issues and also permitgeneral studies of chromosomal domains in interphase nuclei [Trask etal., Hum. Genet., 78:251 (1988)].

Hybridization of pBS21 to a metaphase spread from a cell line withtrisomy 21 is shown in FIG. 4F [protocol II, 4 ng of probe per ul and250 ng of genomic DNA per ul; 3-hr incubation, additional 250 ng ofgenomic DNA per ul (Q=1+1); 3-hr probe incubation and 16-hrhybridization]. A small amount of hybridization is visible near thecentromeres of the other acrocentric chromosomes.

FIG. 4G shows two interphase nuclei from the same hybridization whichclearly show the three chromosome 21 domains. Hybridization with probeprepared according to protocol I resulted in higher relative intensityof the shared signals on the D- and G-group chromosomes, andconsequently it was more difficult to determine the number of number 21chromosomes in interphase (not shown). Increasing stringency by using ahybridization mixture with 55% formamide and 0.15 M NaCl/15 mM sodiumcitrate, which lowers the melting temperature about 8° C., did notreduce the unwanted hybridization. Addition of unlabeled pA ribosomalDNA [Erikson et al., Gene, 16:1 (1981)] also was ineffective atincreasing specificity.

The centromeric region of the D- and G-group chromosomes containribosomal [Erikson et al., id] and alpha satellite sequences and perhapsothers [Choo et al., Nucleic Acids Res., 16: 1273 (1988)]. These arerelatively low copy sequences shared with only a few chromosomes, soProtocol I is not very effective at suppressing them relative to thechromosome 21-specific sequences. In addition, these sequences areclustered on the chromosomes, so that even much reduced hybridization isclearly visible. This is especially distracting in analysis ofinterphase nuclei. Calculations indicate that addition of severalaliquots of freshly denatured genomic DNA periodically during theincubation (protocol II) should increase the staining specificity. FIG.4F shows a protocol II hybridization, using two aliquots of genomic DNA,to a metaphase spread from a trisomy 21 cell line. Intense hybridizationto the three number 21 chromosomes is clearly visible and hybridizationto the other D- and G-group chromosomes has been reduced to anacceptable level. FIG. 4G shows that hybridization to chromosomes otherthan chromosome 21 is sufficiently low that the three chromosome 21domains are clearly visible in interphase nuclei. In practice, the mostconvenient procedure for suppressing the shared acrocentrichybridization might be inclusion of unlabeled DNA from one of the otherD- or G-group chromosome libraries (or unlabeled cloned DNA from justthese sequences, if available) as additional competitor. The use oflibraries from non-target chromosomes as blocker for a probe may ingeneral improve contrast. The specific sequences in the probe will notbe blocked (Q=0) no matter how much competitor for the shared sequencesis added.

VI.C. Hybridization of Yeast Artificial Chromosomes (YACS) to HumanMetaphase Spread

YACS. Seven yeast clones HY1, HY19, HY29, HYA1.A2, HYA3.A2, HYA3.A9, andHYA9.E6 were obtained from D. Burke (Washington University, St. Louis,Mo.). The lengths of the human DNA in the clones ranged from about 100kb to about 600 kb. Gel electrophoresis was performed to verify the sizeof these inserts. Each of these clones was grown up and total DNA wasisolated. The isolated DNA was biotinylated by nick translation so that10-30% of the thyrnidine was replaced by biotin-11-dUTP. Theconcentration of the total labeled DNA after nick translations is in therange of 10-20 ng/ul.

Blocking DNA. Human placental DNA (Sigma) was treated with proteinase Kand extracted with phenol and sonicated to a size range of 200-600 bp.Total DNA isolated from yeast not containing an artificial chromosomewas sonicated to a similar size range. Both of these DNA's weremaintained at a concentration of 1-10 ug/ul.

Fluorescence in situ hybridization (FISH). Hybridization followed theprocedures of Pinkel et al. (1988), supra (as exemplified in Sections Vand VI, supra) with slight modifications. Metaphase spreads wereprepared from methotrexate synchronized cultures according to theprocedures of Harper et al. PNAS (USA) 78: 4458-4460, (1981). Cells werefixed in methanol/acetic acid, fixed (3:1), dropped onto slides, airdried, and stored at −20° C. under nitrogen gas until used. The slideswere then immersed two minutes in 70% formamide/2×SSC to denature thetarget DNA sequences, dehydrated in a 70-85-100% ethanol series, and airdried. (SSC is 0.15 M NaCl/0.015 M′Na Citrate, pH 7). Ten—100 ng ofbiotinylated yeast DNA, and approximately 1 ug each of unlabeled yeastand human genomic DNA were then added to the hybridization mix (finalvolume 10 ul, final composition 50% formamide/2×SSC/10% dextransulfate), heated to 70° C. for 5 min., and then incubated at 37° C. for1 hr to allow the complementary strands of the more highly repeatedsequences to reassociate.

The hybridization mixture was then applied to the slide (approximately 4cm² area) and sealed with rubber cement under a glass cover slip. Afterovernight incubation at 37° C. the coverslip was removed and the slidewashed 3 times 3 min each in 50% formamide/2×SSC at 42-45° C., and oncein PN buffer [mixture of 0.1 M NaH₂PO₄ and 0.1 M Na₂HPO₄ to give pH 8;0.1% Nonidet P-40 (Sigma)]. The bound probe was then detected withalternating 20 min incubations (room temperature in avidin-FITC andgoat-anti-avidin antibody, both at 5 ug/ml in PNM buffer (PN buffer plus5% nonfat dry milk, centrifuged to remove solids; 0.02% Na azide).Avidin and anti-avidin incubation were separated by 3 washes of 3 mineach in PN buffer. Two or three layers of avidin were applied (Avidin,DCS grade, and biotinylated goat-anti-avidin are obtained from VectorLaboratories Inc., Burlingame, Calif.).

FIG. 5 shows the hybridization of HYA3.A2 (580 kb of human DNA) to12q21.1. The location of the hybridization was estabilished by using aconventional fluorescent banding technique employing theDAPI/actinomycin D procedure: Schweizer, “Reverse fluorescent chromosomebanding with chromomycin and DAPI,” Chromosoma, 58:307-324 (1976). Thehybridization signal forms a band across the width of each of thechromosome 12s, indicating the morphology of the packing of DNA in thatregion of the chromosome.

The YAC clone positions are attributed as shown in Table 2 below.

TABLE 2 YAC Competition Hybridization YAC Clone Insert Size LocalizationHY1 120 Xq23 HY19 450 8q23.3 21q21.1 HY29 500 14q12 HYA1.A2 250 6q16HYA3.A2 580 12q21.1 HYA3.A9 600 14q21 HYA9.E6 280 1p36.2 3q22

VI.D. Hybridization With Human/Hamster Hybrid Cell

Essentially the same hybridization and staining conditions were used inthis example as for those detailed in the procedure of Pinkel et al.(1988), supra and exemplified in Sections V.C. and VI.B., supra. In thisexample, 400 ng of biotin labeled DNA from a hamster-human hybrid cellthat contains one copy of human chromosome 19 was mixed with 1.9 ug ofunlabeled human genomic DNA in 10 ul of hybridization mix. Hybridizationwas for approximately 60 hours at 37° C. Fluorescent staining of thebound probe and counterstaining of the chromosomes was as in the otherexamples above. FIG. 6 shows the results of the hybridization.

VII. Specific Applications

The present invention allows microscopic and in some cases flowcytometric detection of genetic abnormalities on a cell by cell basis.The microscopy can be performed entirely by human observers, or includevarious degrees of addititional instrumentation and computationalassistance, up to full automation. The use of instrumentation andautomation for such analyses offers many advantages. Among them are theuse of fluorescent dyes that are invisible to human observers (forexample, infared dyes), and the opportunity to interpret resultsobtained with multiple labeling methods which might not besimultaneously visible (for example, combinations of fluorescent andabsorbing stains, autoradiography, etc.) Quantitative measurements canbe used to detect differences in staining that are not detectable byhuman observers. As is described below, automated analysis can alsoincrease the speed with which cells and chromosomes can be analysed.

The types of cytogenetic abnormalities that can be detected with theprobes of this invention include: Duplication of all or part of achromosome type can be detected as an increase in the number or size ofdistinct hybridization domains in metaphase spreads or interphase nucleifollowing hybridization with a probe for that chromosome type or region,or by an increase in the amount of bound probe. If the probe is detectedby fluorescence, the amount of bound probe can be determined either flowcytometrically or by quantitative fluorescence microscopy. Deletion of awhole chromosome or chromosome region can be detected as a decrease inthe number or size of distinct hybridization domains in metaphasespreads or interphase nuclei following hybridization with a probe forthat chromosome type or region, or by a decrease in the amount of boundprobe. If the probe is detected by fluorescence, the amount bound can bedetermined either flow cytometrically or by quantitative fluorescencemicroscopy. Translocations, dicentrics and inversions can be detected inmetaphase spreads and interphase nuclei by the abnormal juxtaposition ofhybridization domains that are normally separate following hybridizationwith probes that flank or span the region(s) of the chromosome(s) thatare at the point(s) of rearrangement. Translocations involve at leasttwo different chromosome types and result in derivative chromosomespossessing only one centromere each. Dicentrics involve at least twodifferent chromosome types and result in at least one chromosomefragment lacking a centromere and one having two centromeres. Inversionsinvolve a reversal of polarity of a portion of a chromosome.

VII.A Banding Analysis

Substantial effort has been devoted during the past thirty years todevelopment of automated systems (especially computer controlledmicroscopes) for automatic chromosome classification and aberrationdetection by analysis of metaphase spreads. In recent years, effort hasbeen directed at automatic classification of chromosomes which have beenchemically stained to produce distinct banding patterns on the variouschromosome types. These efforts have only partly succeeded because ofthe subtle differences in banding pattern between chromosome types ofapproximately the same size, and because differential contraction ofchromosomes in different metaphase spreads causes a change in the numberand width of the bands visible on chromosomes of each type. The presentinvention overcomes these problems by allowing construction of reagentswhich produce a staining pattern whose spacing, widths and labelingdifferences (for example different colors) are optimized to facilitateautomated chromosome classification and aberration detection. This ispossible because hybridization probes can be selected as desired alongthe lengths of the chromosomes. The size of a band produced by such areagent may range from a single small dot to a substantially uniformcoverage of one or more whole chromosomes. Thus the present inventionallows construction of a hybridization probe and use of labeling means,preferably fluorescence, such that adjacent hybridization domains can bedistinguished, for example by color, so that bands too closely spaced tobe resolved spatially can be detected spectrally (i.e. if red and greenfluorescing bands coalesce, the presence of the two bands can bedetected by the resulting yellow fluorescence).

The present invention also allows construction of banding patternstailored to particular applications. Thus they can be significantlydifferent in spacing and color mixture, for example, on chromosomes thatare similar in general shape and size and which have similar bandingpatterns when conventional techniques are used. The size, shape andlabeling (e.g. color) of the hybridization bands produced by the probesof the present invention can be optimized to eliminate errors in machinescoring so that accurate automated aberration detection becomespossible. This optimized banding pattern will also greatly improvevisual chromosome classification and aberration detection.

The ease of recognition of specific translocation breakpoints can beimproved by using a reagent closely targeted to the region of the break.For example, a high complexity probe of this invention comprisingsequences that hybridize to both sides of the break on a chromosome canbe used. The portion of the probe that binds to one side of the breakcan be detected differently than that which binds to the other, forexample with different colors. In such a pattern, a normal chromosomewould have the different colored hybridization regions next to eachother, and such bands would appear dose together. A break would separatethe probes to different chromosomes or result in chromosomal fragments,and could be visualized as much further apart on an average.

VII.B Biological Dosimetry

One approach to biological dosimetry is to measure frequencies ofstructurally aberrant chromosomes as an indication of the genetic damagesuffered by individuals exposed to potentially toxic agents. Numerousstudies have indicated the increase in structural aberration frequencieswith increasing exposure to ionizing radiation and other agents, whichare called clastogens. Dicentric chromosomes are most commonly scoredbecause their distinctive nature allows them to be scored rapidlywithout banding analysis. Rapid analysis is important because of the lowfrequency of such aberrations in individuals exposed at levels found inworkplaces (˜2×10⁻³/cell). Unfortunately, dicentrics are not stablyretained so the measured dicentric frequency decreases with time afterexposure. Thus low level exposure over long periods of time does notresult in an elevated dicentric frequency because of the continuedclearance of these aberrations. Translocations are better aberrations toscore for such dosimetric studies because they are retained more or lessindefinitely. Thus, assessment of genetic damage can be made at timeslong after exposure. Translocations are not routinely scored forbiological dosimetry because the difficulty of recognizing them makesscoring sufficient cells for dosimetry logistically impossible.

The present invention eliminates this difficulty. Specifically,hybridization with a probe which substantially uniformly stains severalchromosomes (e.g. chromosomes 1, 2, 3 and 4) allows immediatemicroscopic identification in metaphase spreads of structuralaberrations involving these chromosomes. Normal chromosomes appearcompletely stained or unstained by the probe. Derivative chromosomesresulting from translocations between targeted and non-targetedchromosomes are recognized as being only partly stained, FIG. 4D. Suchpartially hybridized chromosomes can be immediately recognized eithervisually in the microscope or in an automated manner using computerassisted microscopy. Discrimination between translocations anddicentrics is facilitated by adding to the probe, sequences found at allof the chromosome centromeres. Detection of the centromeric componentsof the probe with a labeling means, for example color, different fromthat used to detect the rest of the probe elements allows readyidentification of the chromosome centromeres, which in turn facilitatesdiscrimination between dicentrics and translocations. This technologydramatically reduces the scoring effort required with previoustechniques so that it becomes feasible to examine tens of thousands ofmetaphase spreads as required for low level biological dosimetry.

VII.C. Prenatal Diagnosis

The most common aberrations found prenatally are trisomies involvingchromosomes 21 (Down syndrome), 18 (Edward syndrome) and 13 (Patausyndrome) and X0 (Turner syndrome), XXY (Kleinfelter syndrome) and XYYdisease. Structural aberrations also occur. However, they are rare andtheir clinical significance is often uncertain. Thus, the importance ofdetecting these aberrations is questionable. Current techniques forobtaining fetal cells for conventional karyotyping, such as,amniocentesis and chorionic villus biopsy yield hundreds to thousands ofcells for analysis. These are usually grown in culture for 2 to 5 weeksto produce sufficient mitotic cells for cytogenetic analysis. Oncemetaphase spreads are prepared, they are analyzed by conventionalbanding analysis. Such a process can only be carried out by highlyskilled analysts and is time consuming so that the number of analysesthat can be reliably carried out by even the largest cytogeneticslaboratories is only a few thousand per year. As a result, prenatalcytogenetic analysis is usually limited to women whose children are athigh risk for genetic disease (e.g. to women over the age of 35).

The present invention overcomes these difficulties by allowing simple,rapid identification of common numerical chromosome aberrations ininterphase cells with no or minimal cell culture. Specifically, abnormalnumbers of chromosomes 21, 18, 13, X and Y can be detected in interphasenuclei by counting numbers of hybridization domains followinghybridization with probes specific for these chromosomes (or forimportant regions thereof such as 21q22 for Down syndrome). Ahybridization domain is a compact, distinct region over which theintensity of hybridization is high. An increased frequency of cellsshowing three domains (specifically to greater than 10%) for chromosomes21, 18 and 13 indicates the occurrence of Down, Edward and Patausyndromes, respectively. An increase in the number of cells showing asingle X-specific domain and no Y-specific domain followinghybridization with X-specific and Y-specific probes indicates theoccurrence of Turner syndrome. An increase in the frequency showing twoX-specific domains and one Y-specific domain indicates Kleinfeltersyndrome, and increase in the frequency of cells showing one X-specificdomain and two Y-specific domains indicates an XYY fetus. Domaincounting in interphase nuclei can be supplemented (or in some casesreplaced) by measurement of the intensity of hybridization using, forexample, quantitative fluorescence microscopy or flow cytometry, sincethe intensity of hybridization is approximately proportional to thenumber of target chromosomes for which the probe is specific. Numericalaberrations involving several chromosomes can be scored simultaneouslyby detecting the hybridization of the different chromosomes withdifferent labeling means, for example, different colors. Theseaberration detection procedures overcome the need for extensive cellculture required by procedures since all cells in the population can bescored. They eliminate the need for highly skilled analysts because ofthe simple, distinct nature of the hybridization signatures of numericalaberrations. Further, they are well suited to automated aberrationanalysis.

The fact that numerical aberrations can be detected in interphase nucleialso allows cytogenetic analysis of cells that normally cannot bestimulated into mitosis. Specifically, they allow analysis of fetalcells found in maternal peripheral blood. Such a feature is advantageousbecause it eliminates the need for invasive fetal cell sampling such asamniocentesis or chorionic villus biopsy.

As indicated in the Background, the reason such embryo-invasive methodsare necessary is that conventional karyotyping and banding analysisrequires metaphase chromosomes. At this time, there are no acceptedprocedures for culturing fetal cells separated from maternal blood toprovide a population of cells having metaphase chromosomes. In that thestaining reagents of this invention can be employed with interphasenuclei, a non-embryo-invasive method of karyotyping fetal chromosomes isprovided by this invention.

The first step in such a method is to separate fetal cells that havepassed through the placenta or that have been shed by the placenta intothe maternal blood. The incidence of fetal cells in the maternalbloodstream is very low, on the order of 10⁻⁴ to 10^(÷6) cells/ml andquite variable depending on the time of gestation; however,appropriately marked fetal cells may be distinguished from maternalcells and concentrated, for example, with high speed cell sorting.

The presence of cells of a male fetus may be identified by a label, forexample a fluorescent tag, on a chromosome-specific staining reagent forthe Y chromosome. Cells that were apparently either lymphocytes orerythrocyte precursors that were separated from maternal blood wereshown to be Y-chromatin-positive. [Zillacus et al., Scan. J. Haematol,15: 333 (1975); Parks and Herzenberg, Methods in Cell Biology, Vol. 10,pp. 277-295 (Academic Press, N.Y., 1982); and Siebers et al.,Humangenetik, 28: 273 (1975)].

A preferred method of separating fetal cells from maternal blood is theuse of monoclonal antibodies which preferentially have affinity for somecomponent not present upon the maternal blood cells. Fetal cells may bedetected by paternal HLA (human leukocyte antigen) markers or by anantigen on the surface of fetal cells. Preferred immunochemicalprocedures to distinguish between fetal and maternal leukocytes on thebasis of differing HLA type use differences at the HLA-A2, −A3, and −B7loci, and further preferred at the −A2 locus. Further, first and secondtrimester fetal trophoblasts may be marked with antibody against theinternal cellular constituent cytokeratin which is not present inmaternal leukocytes. Exemplary monoclonal antibodies are described inthe following references.

Herzenberg et al., PNAS, 76: 1453 (1979), reports the isolation of fetalcells, apparently of lymphoid origin, from maternal blood byfluorescence activated cell sorting (FACS) wherein the separation wasbased on the detection of labeled antibody probes which bind HLA-A2negative cells in maternal blood. Male fetal cells separated in thatmanner were further identified by quinacrine staining of Y-chromatin.

Covone et al., Lancet, Oct. 13, 1984: 841, reported the recovery offetal trophoblasts from maternal blood by flow cytometry using amonoclonal antibody termed H315. Said monoclonal reportedly identifies aglycoprotein expressed on the surface of the human syncytiotrophoblastas well as other trophoblast cell populations, and that is absent fromperipheral blood cells.

Kawata et al., J. Exp. Med., 160: 653 (1984), discloses a method forisolating placental cell populations from suspensions of human placenta.The method uses coordinate two-color and light-scatter FACS analysis andsorting. Five different cell populations were isolated on the basis ofsize and quantitative differences in the coordinate expression of cellsurface antigens detected by monoclonal antibodies against an HLA-A, B,C monomorphic determinant (MB40.5) and against human trophoblasts(anti-Trop-1 and anti-Trop-2).

Loke and Butterworth, J. Cell Sci., 76: 189 (1985), describe twomonoclonal antibodies, 18B/A5 and 18A/C4, which are reactive with firsttrimester cytotrophoblasts and other fetal epithelial tissues includingsyncytiotrophoblasts.

A preferred monoclonal antibody to separate fetal cells from maternalblood for staining according to this invention is the anti-cytokeratinantibody Cam 5.2, which is commercially available from Becton-Dickinson(Franklin Lakes, N.J., USA).

Other preferred monoclonal antibodies for separating fetal cells frommaternal blood are those disclosed in co-pending, commonly owned U.S.patent application, U.S. Ser. No. 389,224, filed Aug. 3, 1989, entitled“Method for Isolating Fetal Cytotrophoblast Cells”. [See also: in Fisheret al., J. Cell. Biol., 109 (2): 891-902 (1989)]. The monoclonalantibodies disclosed therein react specifically with antigen on firsttrimester human cytotrophoblast cells, which fetal cells have thehighest probability of reaching the maternal circulation. Saidapplication and article are herein specifically incorporated byreference. Briefly, the disclosed monoclonal antibodies were raised byinjection of test animals with cytotrophoblast cells obtained fromsections of the placental bed, that had been isolated by uterineaspiration. Antibodies raised were subjected to several cytologicalscreens to select for those antibodies which react with thecytotrophoblast stem cell layer of first trimester chorionic villi.

Preferred monoclonal antibodies against such first trimestercytotrophoblast cells disclosed by Fisher et al. include monoclonalantibodies produced from the following hybridomas deposited at theAmerican Tyupe Culture Collection (ATCC; Rockville, Md., USA) under theBudapest Treaty:

Hybridoma ATCC Accession # J1D8 HB10096 P1B5 HB10097

Both hybridoma cultures were received by the ATCC on Apr. 4, 1989 andreported viable thereby on Apr. 14, 1989.

Fisher et al. state that fetal cells isolated from maternal blood by useof said monoclonal antibodies are capable of replication in vitro.Therefore, fetal cells isolated by the method of Fisher et al., that is,first trimester fetal cytotrophoblasts, may provide fetal chromosomalmaterial that is both in metaphase and in interphase.

The fetal cells, preferably leukocytes and cytotrophoblasts, morepreferably cytotrophoblasts, once marked with an appropriate antibodyare then separated from the maternal cells either directly or bypreferably separating and concentrating said fetal cells by cell sortingor panning. For example, FACS may be used to separate fluorescentlylabeled fetal cells, or flow cytometry may be used.

The fetal cells once separated from the maternal blood can then bestained according to the methods of this invention with appropriatechromosome-specific staining reagents of this invention, preferablythose of particular importance for prenatal diagnosis. Preferredstaining reagents are those designed to detect aneuploidy, for example,trisomy of any of several chromosomes, including chromosome types 21,18, 13, X and Y and subregions on such chromosomes, such as, subregion21q22 on chromosome 21.

Preferably, a fetal sample for staining analysis according to thisinvention comprises at least 10 cells or nuclei, and more preferablyabout 100 cells or nuclei.

VII.D Tumor Cytogenetics

Numerous studies in recent years have revealed the existence ofstructural and numerical chromosome aberrations that are diagnostic forparticular disease phenotypes and that provide clues to the geneticnature of the disease itself. Prominent examples include the closeassociation between chronic myelogeneous leukemia and a translocationinvolving chromosome 9 and 22, the association of a deletion of aportion of 13q14 with retinoblastoma and the association of atranslocation involving chromosomes 8 and 14 with Burkitts lymphoma.Current progress in elucidating new tumor specific abnormalities islimited by the difficulty of producing representative, high qualitybanded metaphase spreads for cytogenetic analysis. These problems stemfrom the fact that many human tumors are difficult or impossible to growin culture. Thus, obtaining mitotic cells is usually difficult. Even ifthe cells can be grown in culture, there is the significant risk thatthe cells that do grow may not be representative of the tumorigenicpopulation. That difficulty also impedes the application of existinggenetic knowledge to clinical diagnosis and prognosis.

The present invention overcomes these limitations by allowing detectionof specific structural and numerical aberrations in interphase nuclei.These aberrations are detected as described supra. Hybridization withwhole chromosome probes will facilitate identification of previouslyunknown aberrations thereby allowing rapid development of newassociations between aberrations and disease phenotypes. As the geneticnature of specific malignancies becomes increasingly well known, theinterphase assays can be made increasingly specific by selectinghybridization probes targeted to the genetic lesion. Translocations atspecific sites on selected chromosomes can be detected by usinghybridization probes. that closely flank the breakpoints. Use of theseprobes allows diagnosis of these specific disease phenotypes.Translocations may be detected in interphase because they bring togetherhybridization domains that are normally separated, or because theyseparate a hybridization domain into two, well separated domains. Inaddition, they may be used to follow the reduction and reemergence ofthe malignant cells during the course of therapy. Interphase analysis isparticularly important in such a application because of the small numberof cells that may be present and because they may be difficult orimpossible to stimulate into mitosis.

Duplications and deletions, processes involved in gene amplification andloss of heterozygosity, can also be detected in metaphase spreads andinterphase nuclei using the techniques of this invention. Such processesare implicated in an increasing number of different tumors.

VIII. Detection of BCR-ABL Fusion in Chronic Myelogenous Leukemia (CML)

Probes. This section details a CML assay based upon FISH with probesfrom chromosomes 9 and 22 that flank the fused BCR and ABL sequences inessentially all cases of CML (FIG. 8). The BCR and ABL probes used inthe examples of this section were kindly provided by Carol A. Westbrookof the Department of Medicine, Section of Hematology/Oncology at theUniversity of Chicago Medical Center in Chicago, Ill. (USA).

The ABL probe on chromosome 9, c-hu-ABL, is a 35-kb cosmid (pCV105)clone selected to be telomeric to the 200-kb region of ABL between exonsIB and II in which the breaks occur (24). The BCR probe on chromosome22, PEM12, is an 18-kb phage clone (in EMBL3) that contains part of, andextends centromeric to, the 5.8-kb breakpoint duster region of the BCRgene in which almost all CML breakpoints occur. FISH was carried outusing a biotin labeled ABL probe, detected with the fluorochrome Texasred, and a digoxigenin labeled BCR probe, detected with the greenfluorochrome FITC. Hybridization of both probes could be observedsimultaneously using a fluorescence microscope equipped with a doubleband pass filter set (Omega Optical).

FIG. 8 is a schematic representation of the BCR gene on chromosome 22,the ABL gene of chromosome 9, and the BCR-ABL fusion gene on thePhiladelphia chromosome, showing the location of CML breakpoints andtheir relation to the probes. Exons of the BCR gene are depicted assolid boxes. The Roman numeral I refers to the first exon of the BCRgene; the arabic numerals 1-5 refer to the exons within the breakpointcluster region, here indicated by the dashed line. The approximatelocation of the 18 kb phage PEM12 probe (the BCR probe) is indicated bythe open horizontal bar. Since the majority of breakpoints in CML occurbetween exons 2 and 4,15 kb or more of target for PEM12 will remain onthe Philadelphia chromosome. In the classic reciprocal translocation afew kb of target for PEM12 (undetectable fluorescent signal) will befound on the derivative chromosome. The map and exon numbering (not toscale) is adapted from Heisterkamp et al. (ref. 34, supra).

Exons of the ABL gene are depicted as open vertical bars (not to scale).The Roman numerals Ia and Ib refer to the alternative first exons, andII to the second exon. Exon II is approximately 25 kb upstream of theend of the 28 kb cosmid c-hu-abl (the ABL probe). All CML breakpointsoccur upstream of exon II, usually between exons Ib and Ia, within aregion that is approximately 200kb in length. Thus, c-hu-abl will alwaysbe 25 to 200 kb away from the fusion junction. The map (not to scale) isadapted from Heisterkamp et al. (ref. 35, supra). The BCR-ABL fusiongene is depicted. In CML, PEM12 will always lie at the junction, andc-hu-abl will be separated from PEM12 by 25 to 225 kb.

Sample Preparation: CML-4: Peripheral blood was centrifuged for 5 min.Ten drops of interface was diluted with PBS, spun down, fixed inmethanol/acetic acid (3:1), and dropped on slides. CML-2, 3, 7: Five to10 drops of marrow diluted with PBS to prevent clotting were fixed inmethanol/acetic acid and dropped on slides. CML-1, 4, 5, 6: Peripheralblood and/or bone marrow was cultured in RPMI 1640 supplemented with 10%fetal calf serum, an antibiotic mixture (gentamycin 500 mg/ml), and 1%L-glutamine for 24 h. Cultures were synchronized according to J. J.Yunis and M. E. Chandler Prog. in Clin. Path., 7:267 (1977), andchromosome preparations followed Gibis and Jackson, Karyogram, 11:91(1985).

Hybridization and Detection Protocol. Hybridization followed proceduresdescribed by D. Pinkel et al. (27), Trask et al. (25), and J. B.Lawrence et al (30), with modifications. The BCR probe wasnick-translated (Bethesda Research Laboratories Nick-Translation System)with digoxigenin-11-dUTP (Boehringer Mannheim Biochemicals) with anaverage incorporation of 25%. The ABL probe was similarlynick-translated with biotin-11-dUTP (Enzo Diagnostics).

1. Hybridization. Denature target interphase cells and/or metaphasespreads on glass slides at 72° C. in 70% formamide/2×SSC at pH 7 for 2min. Dehydrate in an ethanol series (70%, 85%, and 100% each for 2min.). Air dry and place at 37° C. (2×SSC is 0.3M NaCl/30 mM sodiumcitrate). Heat 10 ml of hybridization mixture containing 2 ng/ml of eachprobe, 50% formamide/2×SSC, 10% dextran sulphate, and 1 mg/ml humangenomic DNA (sonicated to 200-600 bp) to 70° C. for 5 min. to denaturethe DNA. Incubate for 30 min. at 37° C. Place on the warmed slides,cover with a 20 mm×20 mm coverslip, seal with rubber cement, andincubate overnight in a moist chamber at 37° C. Remove coverslips andwash three times for 20 minutes each in 50% formamide/2×SSC pH 7 at 42°C., twice for 20 minutes each in 2×SSC at 42° C., and finally rinse atroom temperature in 4×SSC.

2. Detection of Bound Probes: All incubation steps are performed withapproximately 100 ml of solution at room temperature under coverslips.The biotinylated ABL probe was detected first, then thedigoxigenin-labeled BCR probe.

a. Biotinylated ABL Probe: Preblock with 4×SSC/1% bovine serum albumin(BSA) for 5 min. Apply Texas Red-avidin (Vector Laboratories Inc., 2mg/ml in 4×SSC/1% BSA) for 45 min. Wash in 4×SSC once, 4×SSC/1% Triton-X100 (Sigma) and then again in 4×SSC, 5 min. each. Preblock for 5 min. inPNM (PN containing 5% non-fat dry milk and 0.02% sodium azide andcentrifuged to remove solids. PN is 0.1 M NaH₂PO₄/0.1M Na₂HPO₄, 0.05%NP40, pH 8). Apply biotinylated goat anti-avidin (Vector LaboratoriesInc., 5 mg/ml in PNM) for 45 min. Wash twice in PN for 5 min. Apply asecond layer of Texas Red-avidin (2 mg/ml in PNM) for 45 min. Wash twicein PN for 5 min. each.

b. Digoxigenin-Labeled BCR Probe: Preblock with PNM for 5 min. Applysheep anti-digoxigenin antibody (obtained from D. Pepper, BoehringerMannheim Biochemicals, Indianapolis, Ind.; 15.4 mg/ml in PNM) for 45min. Wash twice in PN for 5 min. each. Preblock with PNM for 5 min.Apply rabbit-anti-sheep antibody conjugated with FITC (OrganonTeknika-Cappel, 1:50 in PNM) for 45 min. Wash twice for 5 min. each inPN. If necessary, the signal is amplified by preblocking for 5 min. withPNM and applying sheep anti-rabbit IgG antibody conjugated to FITC(Organon Teknika-Cappel, 1:50 in PNM) for 45 min. Rinse in PN.

3. Visualization: The slides are mounted fluorescence antifade solution[G. D. Johnson and J. G. Nogueria, J. Immunol. Methods, 43:349 (1981))(ref. 31, supra)] containing 1 mg/ml 4′,6-amidino-2-phenylindole (DAPI)as a counterstain, and examined using a FITC/Texas red double-band passfilter set (Omega Optical) on a Zeiss Axioskop.

The method used for BCR-ABL PCR tested herein was that described inHegewisch-Becker et al. for CML-3, 4 and 7 (ref. 32, supra), and Kohleret al., for CML-5 and 6 (ref. 33, supra).

Results. ABL and BCR hybridization sites were visible on both chromatidsof chromosomes in most metaphase spreads. The ABL probe bound tometaphase spreads from normal individuals (FIG. 9A) near the telomere on9q while the BCR probe bound at 22q11 (FIG. 9B). Hybridization with theABL or BCR probe to normal interphase nuclei typically resulted in twotiny fluorescent dots corresponding to the target sequence on bothchromosome homologues. The spots were apparently randomly distributed inthe two dimensional nuclear images and were usually well separated. Afew cells showed two doublet hybridization signals probably a result ofhybridization to both sister chromatids of both homologues in cellswhich had replicated this region of DNA (i.e., those in the S- or G2-phase of cell cycle). Dual color FISH of the ABL (red) and BCR (green)probes to normal G1 nuclei yielded two red (ABL) and two green (BCR)hybridization signals distributed randomly around the nucleus.

The genetic rearrangement of CML brings the DNA sequences homologous tothe probes together on an abnormal chromosome, usually the Ph¹, andtogether in the interphase nucleus, as illustrated in FIG. 8. Thegenomic distance between the probe binding sites in the fusion genevaries among CML cases, ranging from 25 to 225 kb, but remains the samein all the cells of a single leukemic done. Dual color hybridizationwith ABL and BCR probes to interphase CML cells resulted in one red andone green hybridization signal located at random in the nucleus, and onered-green doublet signal in which the separation between the two colorswas less than 1 micron (or one yellow hybridization signal forhybridization in very close proximity, see FIG. 10). The randomlylocated red and green signals are ascribed to hybridization to the ABLand BCR genes on the normal chromosomes, and the red-green doubletsignal to hybridization to the BCR-ABL fusion gene. Interphase mappingstudies suggest that DNA sequences separated by less than 250 kb shouldbe separated in interphase nuclei by less than 1 micron (25). As aresult, cells showing red and green hybridization signals separated bygreater than 1 micron were scored as normal since this is consistentwith the hybridization sites being on different chromosomes. However,due to statistical considerations, some normal cells will have red andgreen dots dose enough together to be scored as abnormal. In these twodimensional nuclear analyses, 9 out of 750 normal nuclei had red andgreen hybridization signals less than 1 micron of each other. Thus,approximately 1% of normal cells were classified as abnormal.

Table 3 shows the hybridization results for 7 samples from 6 CML casesalong with conventional karyotypes, and other diagnostic results (PCRand Southern blot data ). All six cases, induding 3 that were found tobe Ph¹ negative by banding analysis (CML-5,-6 and -7), showed red-greenhybridization signals separated by less than 1 micron in greater than50% of nuclei examined. In most, the fusion event was visible in almostevery cell. One case (CML-7) showed fusion signals in almost every celleven though PCR analysis failed to detect the presence of a fusion geneand banding analysis did not reveal a Philadelphia chromosome.

Hybridization to metaphase spreads was performed in three cases(CML-1,-5 and -6). All of these showed red and green hybridizationsignals in close proximity on a single acrocentric chromosome. In twocases, scored as t(9:22)(q34;q11) by banding, the red-green pair was inclose proximity to the telomere of the long arm of a small acrocentricchromosome as expected for the Ph¹ (FIG. 9C). One case (CML-6) wassuspected by classical cytogenetics to have an insertion of chromosomalmaterial at 22q11. Dual color hybridization to metaphase spreads fromthis case showed the red-green pair to be centrally located in a smallchromosome (FIG. 9D). That result is consistent with formation of theBCR-ABL fusion gene by an insertion. In one case (CML-1), two pairs ofred-green doublet signals were seen in 3 out 150 (2%) interphase nuclei.That may indicate a double Ph¹ (or double fusion gene) in those cells.Such an event was not detected by standard cytogenetics, which waslimited to analysis of 25 metaphase spreads. The acquisition of anadditional Ph¹ is the most frequent cytogenetic event accompanying blasttransformation, and its cytogenetic detection may herald diseaseacceleration.

Simultaneous hybridization with ABL and BCR probes to metaphase spreadsof the CML derived cell line K-562 showed multiple red-greenhybridization sites along both arms of a single acrocentric chromosome.Hybridization to interphase nuclei showed that the red and green signalswere confined to the same region of the nucleus. That is consistent withtheir being localized on a single chromosome. Twelve to fifteenhybridization pairs were seen in each nucleus indicating correspondingamplification of the BCR-ABL fusion gene (see FIGS. 9E and 9F). Thesefindings are consistent with previous Southern blot data showingamplification of the fusion gene in this cell line (26).

In summary, analysis of interphase cells for seven CML, and four normalcell samples using dual color FISH with ABL and BCR probes suggests theutility of this approach for routine diagnosis of CML and clinicalmonitoring of the disease. Among its very important advantages are theability to obtain genetic information from individual interphase ormetaphase cells in less than 24 hours. Thus, it can be applied to allcells of a population, not just to those that fortuitously or throughculture, happen to be in metaphase. Further, the genotypic analysis canbe associated with cell phenotype, as judged by morphology or othermarkers, thereby permitting the study of lineage specificity of cellscarrying the CML genotype as well as assessment of the frequency ofcells carrying the abnormality.

Random juxtaposition of red and green signals in two dimensional imagesof normal cells, which occurs in about 0.01 of normal cells, sets thelow frequency detection limit. That detection limit may be lowered bymore complete quantitative measurement of the separation and intensityof the hybridization signals in each nucleus using computerized imageanalysis. Such analysis will be particularly important in studyingpatient populations in which the cells carrying the BCR-ABL fusion atlow frequency (e.g., during remission, after bone marrowtransplantation, during relapse or in model systems).

This assay also should be advantageous for detection of CML cells duringtherapy when the number of cells available for analysis is low sinceonly a few cells are required. Finally, simple counting of hybridizationspots allows for the detection and quantitative analysis ofamplification of the BCR-ABL fusion gene as illustrated for the K562cell line (FIG. 9E). Quantitative measurement of fluorescence intensitymay assist with such an analysis.

IX. Detection of the Retinoblastoma Gene in Metaphase Chromosomes andInterphase Nuclei

Probes. Fourteen lambda phage clones which form three contigs(overlapping nucleic acid sequences) and span all the exons of the Rb-1gene were obtained from Yuen Kai Fung [Division of Hematology andOncology, Childrens' Hospital of Los Angeles, University of SouthernCalifornia, Los Angeles, Calif. 90027 (USA)]. The phage DNA was labeledeither by biotin-dUTP or digoxigenin-dUTP using the Bio Nick™ LabelingSystem [BRL Life Technologies, Inc., Gaithersburg, Md. (USA)].

A probe specific to the 13/21 centromeric alphoid repetitive sequencewas used in assisting identification of chromosome 13 in metaphasepreparations. The 13/21 centromeric probe was prepared by the polymerasechain reaction (PCR) according to methods detailed in Weier et al., Hum.Genet. 87(4):489-494 (1991). Briefly, the probe was made by PCR usingflow sorted human chromsome 21s as a template and two primers (30 μM)specific for the alphoid sequence. The product was labeled during thePCR reaction by including biotin-11-dUTP (100%). Oligonucleotide primersused were W21R1 (5′-GGATAGCTTAACGATTTCGTTGGAAAC-3′) and W21R2(5′-CAAACGTGCTCAAAGTAAGGGAATG-3′). They were synthesized usingphosphoramidite chemistry on a DNA synthesizer (Applied Biophysics,Foster City, Calif., model 380B). Synthesis and further purification ofthe oligonucleotides by C18 reverse phase chromatography and HPLC wasperformed according to the specifications of the manufacturer (WatersChromatography, Milford, Mass., USA). Using the flow sorted chromosomalDNA as a template these primers generate a 135 bp product.

Cell samples. PHA-stimulated normal peripheral blood lymphocytes,cultured human skin fibroblasts, two fibroblast cell lines fromretinoblastoma patients GM05877 46, XX, del(13) (pter-q14.1::q21.2-qter)and GM01142A 46, XX, del(13) (pter-q14.1::q22.1-qter) obtained from theNIGMS (National Institute of General Medical Science) Human GeneticMutant Cell Repository (Camden, N.J.), and clinical human breast cancersamples obtained either by fine-needle aspiration or disaggregation offresh tumor tissue were used [made available by Fred Waldman, M.D.,Department of Laboratory Medicine, University of California, SanFrancisco, Calif. (USA)]. Cell lines were either treated with colcemidto prepare metaphases or grown to confluency to obtain G0/G1 interphasenuclei. All samples were fixed in 3:1 methanol-acetic acid and droppedon microscope slides. Before in situ hybridization the slides weretreated with (1 ug/50 ml) Proteinase K [Boehringer Mannheim GmbH,Indianapolis, Ind.) (USA) for 7.5 min at 37° C.

In situ hybridization. FISH was done using modifications of previouslypublished methods (Pinkel et al., 1986; Trask et al. 1989). Thehybridization mixture consisting of 20-40 ng of labeled probe, 5-10 μgunlabeled human placental DNA in 50% formamide, 2×SSC and 10% dextransulphate was denaturated for 5 min at 70° C. and then allowed toreanneal for 30-60 min at 37° C. In dual color hybridizations, 20 ng ofRb 3′ end probe and 20 ng of Rb 5′ end probe was used. Incohybridizations with the 13/21 centromeric probe, 2 ng of thecentromeric probe was used with 20-40 ng of the Rb-1 probe. The slideswere denatured in 70% formamide, 2×SSC at 70° C. for 2 min.Hybridization was done under a coverslip in a moist chamber at 37° C.for 2 days.

Staining. Briefly, the slides were washed three times in 50% formamide,2×SSC for 10 min, twice in 2×SSC and once in 0.1×SSC at 45° C.Biotin-labeled specimens were stained with (5 ug/ml) FITC- or TexasRed-Avidin [Vector Laboratories, Inc., Burlingame, Calif. (USA)] in4×SSC/1% BSA for 30 minutes at room temperature. Anti-avidin (VectorLaboratories, Inc. (5 ug/ml) incubation was done in PNM buffer for 20min followed by a second layer of FITC/Texas Red-Avidin in PNM buffer.Digoxigenin-labeled probes were detected using an FITC-labeled sheepantibody against digoxigenin and a second layer of rabbit anti-sheepFITC antibody (Vector Laboratories, Inc., Burlingame, Calif.). Beforeeach antibody or avidin treatment the slides were preblocked with either1%BSA or PNM. Between the antibody incubations the slides were washedtwice in 4×SSC and once in 4×SSC/0.1%=TRITON X-100 or three times in PNbuffer. Nuclei were counter-stained with 0.2 ug/ml propidium iodide or0.27 uM DAPI in an antifade solution.

Fluorescence microscopy and digital image analysis. A Nikon fluorescencemicroscope was used in most of the analyses. For interphase analysis atleast 150 nuclei were scored from each sample. To map the metaphasehybridization signals accurately digital image analysis was used. Themulticolor images were stored on computer magnetic disks at anapproximate resolution of 19 pixels/em and analyzed using a specificsoftware program based on the TCL-Image software package. The programdefines the contour of the DAPI-stained chromosome and draws thelongitudinal axis of the chromosome. The hybridization signals are thenoverlaid in pseudocolors on the chromosome image to calculate theirrelative position in terms of the distance from the p-telomere comparedto the total chromosome length (=fractional length scale).

Results. Using fourteen lambda phage clones together (Rb-1 probe), abright and specific hybridization signal on lymphocyte metaphasepreparations in the mid-region of the q-area of chromosome 13 wereobtained (FIGS. 13A and 13B). A more accurate localization of the Rb-1gene was achieved by digital image analysis. The mean distance of theRb-1 signal from the 13 pter (p-terminus) was determined to becompatible with the location of Rb-1 gene in the band q14.2 (FIG. 13C).Analysis of the Rb-1 hybridization from interphase nuclei was firstattempted in normal lymphocytes and fibroblasts (FIG. 13D). Twohybridization signals representing the two gene alleles were detected inabout 90% of the nuclei (FIG. 14A). The remaining 10% showed either oneor three fluorescence signals. In interphase, the fluorescence signalwas not always singular but could appear as 2-4 small adjacentindividual spots probably because the Rb-1 probe consists of threeseparate contigs.

Two fibroblast cell lines from retinoblastoma patients with homozygousdeletions affecting the Rb-1 region were used to test the sensitivity ofFISH in detecting deletions using an absence of a hybridization signalas an indicator of deletions. In both cell lines one Rb-1 signal wasdetected in about 70-80% of the interphase nuclei (FIGS. 13G and 14B).Metaphase preparations from those cell lines hybridized simultaneouslywith the Rb-1 and the 13/21 centromeric probes showing that the normalchromosome 13 had both the centromeric and the Rb-1 signals, whereas theother slightly shortened chromosome 13 hybridized only with thecentromeric probe (FIGS. 13E and 13F).

The Rb-1 probe was also used to study fine needle aspirations and touchpreparations from different breast cancer patients. Although the breastcancer samples had more non-specific background fluorescence thancultured cells, it was still possible to evaluate Rb-1 gene copy numbersfrom individual tumor nuclei (FIG. 13H). As shown in Table 4 below,marked genetic heterogeneity both within and between breast tumors wasfound in the analysis of six cases. The modal Rb-1 gene copy numbervaried from 1-3 in the tumors. As compared to experiments with cellcultures, the clinical samples showed a higher percentage of cellswithout any Rb-1 signals. Table 4 shows the percentage of nucleiexhibiting a defined number of Rb-1 signals/nucleus in six clinicalbreast cancer specimens. The results represent the mean of 2-3hybridization experiments. At least 150 cells were scored from eachslide.

TABLE 4 No. signals/nuclei (%) Tumor 0 1 2 3 4 5 6 DI B156 22 23 45 5 50 0 1.50 B245 28 36 31 3 2 0 0 1.35 B249 2 10 23 36 25 3 0.5 1.82 B25221 14 43 14 3 3 1 1.87 B259 36 49 12 2 1 0 0 1.64 B263 16 3 11 33 20 106 2.25 DI = DNA index

To detect subregions of the Rb-1 gene, single phage clones spanning only8-20 kb of the 200 kb Rb-1 gene were used as hybridization probes. Thehybridization signals from such probes could be seen both in metaphasechromosomes and interphase nuclei, but the hybridization efficiency wassignificantly less than with the pooled Rb-1 probe. In contrast, if 2-5contiguous phage clones were pooled the hybridization was more efficientand more easily evaluated. This approach was used to visualize the 3′and 5′ ends of the Rb-1 gene in interphase nuclei with differentlylabeled probes in a dual-color hybridization (FIG. 13I).

Thus, in conclusion, the Rb-1 gene was mapped to 13q14 by FISH anddigital image analysis confirming the location of the gene to be inclose proximity to the esterase D locus (Sparkes et al., 1980). Alsoshown in this section is that the methods of this invention can be usedto detect deletions involving the Rb-1 locus. In order to verify thepresence of a deletion from unbanded propidium iodide stained metaphasepreparations, it was necessary to use a reference probe which in therepresentative example of this section was a 13/21 pericentromeric alphasatellite probe. Chromosome 13s with a deletion in the Rb-1 locus werethereby identified.

The representative examples of this section demonstrate thatchromosome-specific staining can be used to detect Rb-1 gene deletionsfrom interphase nuclei of cultured fibroblasts from retinoblastomapatients known to have a constitutive deletion in 13q. The usefulness ofchromosome-specific staining is determined by the hybridizationefficiency obtained, which in turn experientially has been found to bedependent on probe target size. Previous studies on interphase FISH havemainly been done using probes to pericentromeric repetitive sequenceswith a target size of a few megabases with hybridization efficienciesaround 90-95% (Pinkel et al. 1986). In experiments using the 150 kb Rb-1probe with cultured cells, the hybridization efficiency obtained ininterphase was about 80-90%, whereas in clinical samples the efficiencywas apparently less since a number of cells exhibited no hybridizationsignals. Poor hybridization efficiency might therefore lead tomisinterpretation of a deletion. Further, in solid tumors having verycomplex karyotypic abnormalities, the distinction between numericalchromosome aberrations and structural abnormalities may be difficult toevaluate. Therefore, it is preferred in analyzing large numbers of solidtumors to co-hybridize with other reference probes for the samechromosome to control for the hybridization efficiency as well as forthe presence of numerical chromosomal abnormalities. The centromeric13/21 alpha satellite probe used successfully in metaphase preparationscannot be applied to interphase analysis because the signals forchromosome 13 and 21 cannot be distinguished. Therefore, for interphaseanalysis, it is preferred that a reference probe specific for chromosome13 be used.

The studies on clinical breast cancer material described in this sectiondemonstrate the genetic heterogeneity of breast cancer. The evaluationof this heterogeneity coupled with the possibility of studying gene copynumbers from morphologically defined individual tumor cells are majoradvantages of the chromosome-specific staining methods of thisinvention.

X. Detection of Chromosome 3 and 17 Aberrations Associated with Cancer

Probes. Two centromeric-specific alpha satellite probes are used in therepresentative examples of this section; one is specific to chromosome17, and the other to chromosome 3. The centromeric-specific probes wereprepared similarly as the 13/21 specific centromeric probes were, asindicated above in Section IX. Specifically, those probes were preparedusing a polymerase chain reaction (PCR) process employing a thermostableenzyme [Saiki et al., Science, 239:487-491 (1988)] as follows.

Probe specific for alpha satellite centromeric repeats on humanchromosome 17. Approximately 50 ng (nanograms) of DNA isolated from theBluescribe plasmid library for chromosome 17 (pBS17) were used as theDNA template. Pinkel et al., PNAS USA, 85:9138-9142 (December 1988)describes the preparation of such Bluescribe libraries as subcloning anentire chromosome 17 library, which is publicly available as LL17NS01 orLA17NS03 [Van Dilla et al., Bio/Technology, 4:537-552 (June 1986)] intoBluescribe plasmids [Statagene, La Jolla, Calif. (USA)].

The reaction buffer consisted of 5 units of Thermus aquaticus (Taq) DNApolymerase [Bethesda Research Laboratories, Gaithersburg, Md. (USA)];mixed with 100 μl amplification/biotinylation buffer [10 mM Tris-HCl, pH8.4 at 20° C; 1.5 mM MgCl₂; 5 mM KCl; and 0.2 mM each of2′-deoxyadenosine 5′-triphosphate (dATP), 2′-deoxyguanosine5′-triphosphate (dGTP), and biuotin-II-dUTP [all the deoxynucleotidetriphosphates were from Sigma, St. Louis, Mo. (USA)]; and 1.2 μM each ofthe two primers WA1 and WA2 [WA1 5′-GAAGCTTA(A/T′(C/G)T(C/A)ACAGAGTT(G/T)AA-3′ and WA2 5′-GCTGCAGATC(A/C)C(A/C)AAG(A/T/C) AGTTTC-3′].Mineral oil (100 μl) [Squibb, Princeton, N.J. (USA)] was layered on topof the reaction mixture to prevent evaporation during the PCR.

DNA amplification and simultaneous biotinylation was performed during 45cycles using an automated thermal cycling system [Weier and Gray, DNA7:441-447 (1988)]. Each cycle began with a thermal denaturation step of90 seconds at 94° C. (120 seconds for the initial denaturation). Primerannealing during the second step of each cycle was performed at 53° C.for 90 seconds. The temperature was then increased slowly (7° C./minute)to 72° C. The cycle was completed by holding that temperature for 120seconds for primer extension. Amplification of alpha satellite DNA wasconfirmed visually by electrophoresis of 5 μl aliquots of the PCRreaction mixture on 4% agarose gels (BRL) in 40 mM Tris-acetate, 1 mMEDTA, pH 8.0 containing 0.5 μ/ml ethidium bromide [Maniatis et al.,(1986), supra]. The concentration of double stranded DNA in the reactionwas determined to be 229 μg/ml by Hoeschst 33258 fluorescence using a TK100 fluorometer [Hoefer Scientific, San Francisco, Calif. (USA)].

Probe specific for alpha satellite centromeric repeats on humanchromosome 3. In vitro DNA amplification was performed usingapproximately 80 ng of CsC1 gradient isolated DNA from the Bluescribeplasmid library for chromosome 3 (pBS3) (400 μg/l) as amplificationtemplate per 200 μl reaction mixture. The reaction buffer was the sameas that used to prepare the chromosome 17 centromeric-specific probeabove except that dTTP is used instead of biotin-11-dUTP.

PCR was performed for 30 cycles using an automated thermal cycler[Perkin-Elmer/Cetus, Norwalk, Conn. (USA)]. The DNA template wasdenatured at 94° C. for 1 minute (1 minute 30 seconds during the firstcycle). Primer annealing and extension were performed at 53° C. and 72°C., respectively. Probe biotinylation and further amplification wasaccomplished in a second reaction by adding a 5 μl aliquot of theproduct to 200 μl reaction mix containing 0.25 mM Biotin-11-dUTP (SigmaSt. Louis, Mo.) in the absence of dTTP and 10 units of Taq polymerase[Weier et al., J. Histochem. Cytochem, 38:421-426 (1990)]. Theamplification/biotinylation reaction was performed during an additional20 PCR cycles. Amplification of degenerate alpha satellite DNA wasconfirmed visually by gel electrophoresis of 10 μl aliquots of the PCRreaction in either 1.8% or 4% agarose (BRL) in 40 mM Tris-acetate, 1 mMEDTA buffer, pH 8.0 containing 0.5 μg/ml ethidium bromide. Aftercompletion of PCR, labeled probe and amplified DNA were stored at −18°C.

Chromosome 3 alpha satellite centromeric-specific repetitive probe.Another chromosome 3 centromeric-specific probe called palpha 3-5 wasobtained from Huntington Willard, Ph.D. [Department of Genetics,Stanford University School of Medicine, Stanford, Calif. (USA)]. Thatprobe was described at the Ninth International Workshop on Human GeneMapping in Paris [Cytogenet. Cell Genet., 46 (14):424, 564 and 712(1987), and 51 (14):111 (1989)]; and a similar probe is described inWaye and Willard, Chromosoma (Berl), 97:475-480 (1989). The palpha 3-5probe was labeled with AAF according to conventional methodology for usein the experiments described below.

3p cosmid probe. A 3p cosmid probe called cCI3-787 was obtained fromYusuke Nakamura, MD, Ph.D [Division of Biochemistry, Cancer Institute,Toshima, Tokyo, 170, Japan]. Its isolation and mapping to 3p21.2-p21.1is described in Yamakawa et al., Genomics, 9(3):536-543 (1991). Thatprobe was amplified and labeled with biotin according to a PCRlinker/adapter method described in Johnson, Genomics, 6:243-251 (1990)and Saunders et al., Nucl. Acids. Res., 17 (22):9027-9037 (1989).

3q cosmid probe. A 3q cosmid probe named J14R1A12 was developed andprovided by Wen-Lin Kuo [Biomedical Department, P.O. Box 5507 (L-452),Lawrence Livermore National Laboratory, Livermore, Calif. 94550 (USA)].It was obtained from a chromosome 21 flow sorted library prepared byconventional means. It was mapped to the location 3q26 using anextrapolation of the fractional length for the probe to a chromosome 3ideogram. It was labeled with biotin-dUTP using the Bio Nick™ LabelingSystem [BRL Life Technologies, Inc. Gaithersburg, Md. (USA)].

Composite Whole Chromosome 3-Specific Probe. A probe specific for thewhole chromosome 3 was a Bluescribe plasmid library for chromosome 3prepared according to Pinkel et al., PNAS USA, 85:9138-9142 (December1988) and named pBS3.

Cell Samples. Used in the following examples were PHA-stimulated normalperipheral blood lymphocytes; two ovarian cancer cell lines, designatedas RMUG-S and RMUG-L, provided by Shiro Nozawa, Md., Ph.D. (Departmentof Obstetrics/Gynecology, School of Medicine, Keio University, 35Shinano-machi, Shinjuku-ku, Tokyo, 160, Japan) and described in Sakayoriet al., Human Cell, 3 (1):52-56 (1990); and a uterine cervicaladenocarcinoma cell line, named TMCC-1, described in Sakamoto, J. TokyoMed. College, 46 (5):925-936 (1988). RMUG-S is a hypodiploid cancer cellline, whereas RMUG-L is a hypertriploid cancer cell line. Both lineswere cloned from the same clinical specimen.

In situ hybridization and staining. The protocols resulting in thehybridizations shown in FIGS. 15, 16 and 17 were the same as those usedin Section IX except that for FIG. 15, the hybridization mixturecontained 5 μg of herring sperm DNA instead of 5 μg of unlabeledplacental DNA, and reannealing of the denatured probe just before insitu hybridization was not performed: and for FIG. 16, the hybridizationmixture contained a reduced amount of 0,5 μg of unlabeled humanplacental DNA explicitly 0.5 μg.

For the results shown in FIGS. 18 and 19, the hybridization protocolsdiffered from that detailed in Section IX in that before hybridization,the slides were pretreated with 100 μg/ml of RNase for 30 minutes at 37°C. and then treated with 1μg/ml of Proteinaise K for 7.5 minutes at 37°C. The hybridization mixture comprised 1 μl biotinylated 3p cosmid(wherein the PCR product was diluted 1:10 with double distilled,deionized water) or 30 ng-40 ng 3q cosmid; 2 ng chromosome 3 alphasatellite centromeric-specific probe (palpha 3-5) labeled with AAF; 5μg-10 μg unlabeled human placental DNA; and 7.0 μl of master mix [whichconsists of 5 ml formamide to which 1 g dextran sulphate and 1 ml 20×SSC(prepared using deionized, double distilled water) was added, the pH ofwhich was adjusted to 7.0 with 1N HCl and the final volume to 7 ml wascompleted with deionized, double distilled water]. The master mixture isstored at −20° C. indefinitely.

For the results shown in FIGS. 18 and 19, a dual color staining protocolwas performed essentially according to Trask and Pinkel, Methods in CellBiology, 33:383400 (1991). Briefly, the slides were washed three timesin the washing solutions for 10 minutes each at 45° C., 2×SSC for 10minutes at 45° C., 0.1×SSC for 10 minutes, and PN buffer (wherein thepercentage of NP-40 is 0.05% rather than 0.1%) for 5 minutes at roomtemperature. The washing solutions comprise 50% formamide:2×SSC (75 mlformamide; 15 ml 20×SSC; and 60 ml deionized, double distilled waterwherein the pH is adjusted to 7.0 with 1 N HCl).

The slides are preblocked with 20 μl PNM buffer under a 22×22 mmcoverslip at room temperature for 5 minutes inside a dark moist chamber.The coverslip is then taken off, and the PNM buffer is drained from theslide.

Twenty μl of anti-AAF and Avidin-Texas Red solution are added to thecells'area per slide and then covered with a 22×22 mm coverslip. Theslides are incubated within a dark moist chamber for 1 hour at roomtemperature. The anti-AAF and Avidin-Texas Red solution is prepared byadding 8 μl of 0.25 μg/μl Avidin-Texas Red (Vector) to 1 ml of thesupernatant of anti-AAF producing mouse cells.

The coverslips are then removed, and the slides are washed withintermittent shaking in the PN buffer thrice for 10 minutes each in adark place.

The cells are then preblocked as described above. Twenty μl ofgoat-anti-mouse-FITC antibody and biotinylated anti-Avidin antibodysolution is added to the cells' area on each slide. The cells arecovered with a coverslip and incubated inside a dark moist chamber forone hour at room temperature. The antibody solution per ml comprises 20μl of goat-anti-mouse-FITC antibody [from Cal Taq (Burlingame, Calif.)i.e., the final concentration is 20 μg/ml] and 10 μl biotinylatedanti-avidin antibody solution at the concentration of 0.5 mg/ml (fromVector Laboratories, i.e., the final concentration is 5 μg/ml) to 970 μlantibody dilution buffer [IX Dulbecco's PBS (Ca, Mg free), 0.05% TWEEN20, 2% normal goat serum].

The coverslips were removed from the slides, and the slides are thenwashed with intermittent shaking in the PN buffer thrice for 10 minuteseach in a dark place. The cells are then preblocked as indicated above.

Twenty μl of the Avidin-Texas Red solution were added to the cells' areaper slide, and then a coverslip was applied. The slides are thenincubated inside a dark moist chamber for one hour at room temperature.The Avidin-Texas Red Solution comprises 8 μl of a 0.25 μg/μlAvidin-Texas Red (Vector) to 1 ml of the antibody dilution buffer.

The coverslips are then removed from the slides, and the slides arewashed in the PN buffer thrice for 10 minutes each in a dark place.About 8 μl of 0.8 μm DAPI (counterstain) in an anti-fade solution isprepared according to J. Immuno. Methods, 43:349 (1981) [100 mgp-phenylene-diamine dihydrochloride (Sigma P1519) in 10 ml Dulbecco'sPBS; pH adjusted to 8 with 0.5 M carbonate-bicarbonate buffer; 90 mlglycerol added; filtered through 0.22 μm; stored at −20° C.] was addedto the slides. The slides mounted with the anti-fade solution can bestored in a dark chamber at 4° C.

Results. FIG. 15A shows the hybridization of the chromosome 17centromere-specific alpha satellite probe to normal lymphocytes whereinin metaphase chromosomes, two bright signals are seen, and in interphasenuclei, two bright, tight hybridization domains are visible. FIG. 15Bshows the hybridization of that probe to the human ovarian mucinouscysto-adenocarcinoma (RMUG-L), wherein in both metaphase and interphase,four signals are visible.

These examples are representative of the use of chromosome-specificrepeat probes for the detection of numerical chromosome aberrations onchromosome 17 which are used as a component of the high complexitystaining probes of this invention.

FIGS. 16A and B show hybridization of the whole chromosome compositeprobe for chromosome 3 (pBS3) (A) to normal lymphocytes and (B) to theovarian cancer cell line (RMUG-L). Two normal chromosome 3s are seen inFIG. 16A, whereas four chromosome 3s are seen in the ovarian cancer cellline (FIG. 16B), of which two are apparently shorter than the intactchromosome 3s, a pattern which is congruent with a 3p deletion in thekaryotype.

Chromosome-specific recombinant lambda libraries have been constructedfor all the human chromosomes by the National Laboratory Gene LibraryProject [Van Dilla et al. (1986), supra]. Subsequently, those librarieswere subcloned into Bluescribe plasmid vectors (Stratagene), and wholechromosome composite probes were generated from the DNA extracted fromthose plasmids [Fuscoe et al., Genomics, 5:100-109 (1989); Collins etal., Genomics 11(4):997-1006(1991)]. Staining with such whole chromosomecomposite probes can be used to detect not only large deletions but alsosubtle translocations and to identify the origin of marker chromosomes.

FIGS. 17A and B (as well as FIGS. 9, 10 and 13, among others) provideexamples of the use of locus-specific probes to count the copy number ofspecific genes in tumor cells and to detect changes in patterns ofhybridization domains. FIGS. 17A and B provide representative examplesof the use of locus-specific probes to detect translocations. Asindicated above, such examples are the first step in locating exactinformation on genetic rearrangements within a locus. The 3q cosmidprobe employed in these studies is just one of many potential probesfrom chromosome 3 that can be used [Yamakawa et al., Genomics,9(3):536-543 (1991)]. Preferably, in metaphase spreads probes withdifferent labels according to their order in a normal chromosome 3 maybe used to detect any structural chromosomal aberrations differing fromthe standard. Further, in either metaphase spreads or interphase nuclei,probes with different labels according to their location in normalchromosomes may be used to detect structural chromosomal aberrations,for example, commonly deleted lesions found in cancer cells.

FIGS. 17A and B show the hybridization of a chromosome 3centromeric-specific alpha satellite repeat probe (the one generated bythe PCR process with the primers WA1 and WA2) and a 3q cosmid probe(J14R1A12 mapped to 3q 26) to, respectively (A) normal lymphocytes and(B) a uterine cervical adenocarcinoma cell line (TMCC-1). As indicatedin the description of the figures above, two pairs of normal chromosome3s are illustrated in (A) whereas a a pair of cosmid signals specific tochromosome region 3q was found to be translocated to another chromosome.

FIGS. 18A and 18B show dual color hybridization to normal lymphocytes,metaphase spread and interphase nucleus, respectively. FIGS. 19A and 19Bshow comparable hybridizations to an ovarian cancer cell line. Achromosome 3 centromeric-specific repetitive probe (AAF labeled palpha3-5 from Huntington Willard) and 3p region-specific (3p21.2 p21.1)cosmid probe (cCI3-787) (that is biotinylated and amplified and labeledby linker adapter PCR) were employed in such hybridizations. Two hundredinterphase nuclei were scored for each experiment.

In FIG. 18A, the image of chromosome 3 from normal lymphocytes wasdigitized by the digital fluorescent microscope and shows one chromosome3 centromeric-specific green signal and one pair of chromosome 3pregion-specific red signal for each chromatid were visible.

In FIG. 18B, the picture of an interphase nucleus from normallymphocytes, taken with a conventional fluorescent microscope, shows twogreenish hybridization domains for the centromeric specific probes andtwo reddish domains for the 3p probe. It was commonly observed that apair of cosmid signals on both chromatids of one chromosome 3 fuses intoa single spot in interphase nuclei.

FIG. 19A shows a partial metaphase spread, one chromosome 3 shows anormal hybridization pattern whereas the other shows a 3p deletion. FIG.19B shows in interphase nuclei, four large greenish domains for thecentromeric probe and two small reddish hybridization domains for the 3pprobe, indicating aneuploidy of chromosome 3, wherein two out of four ofthe chromosome 3s have a 3p deletion.

Eighty-six percent of the interphase nuclei of the normal lymphocytesshowed normal pattern of two green centromeric signals and two redsignals of the 3p cosmid probe. However 98% (94%) of interphase nucleiof the RMUG-S (RMUG-L) cells showed a lesser number of red signals (3pcosmid) than green signals (centromere) suggesting a chromosome 3pdeletion in those cell lines. Among those nuclei, 53% (52%) ofinterphase nuclei of the RMUG-S (RMUG-L) cells showed two domines of the3p cosmid signal and 4 domains of the chromosome 3 centromeric-specificsignals.

FIGS. 20 and 21 show the results of simultaneous hybridizations of anAAF-labeled chromosome 3 centromere-specific probe (from H. Willard) anda biotinylated chromosome 3q cosmid probe (J14R1A12) wherein in FIG. 20the target is a metaphase spread and interphase nucleus of normallymphocytes and wherein in FIG. 21 the target is an interphase nucleusfrom the ovarian cancer cell line (RMUG-S). A pattern for a normalchromosomal complement is shown in FIG. 20 as two chromosome 3centromere-specific green signals and two pairs of chromosome 3q cosmidred signals per cell. An abnormal pattern is shown in FIG. 21 as fourchromosome 3 centromere-specific green signals and four chromosome 3qcosmid red signals, indicating that the cell contains four long arms ofchromosome 3. The results shown in FIGS. 20 and 21 support thefeasibility of detecting 3p deletions in interphase nuclei of tumorcells if combined with the findings of domain number for 3p cosmidsignals. Such a methodology can be applied to detect 3p deletions inclinical tumor specimens for basic research on tumorigenesis andprogression, and adjunctive diagnosis of cancers associated with 3pdeletions, such as, small cell lung cancer, renal cell cancer andovarian cancer.

The descriptions of the foregoing embodiments of the invention have beenpresented for purpose of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to best explain the principles of the invention andits practical application to thereby enable others skilled in the art tobest utilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated. It isintended that the scope of the invention be defined by the claimsappended hereto. All references cited herein are hereby incorporated byreference.

What is claimed is:
 1. A method of staining targeted interphase chromosomal material based upon a nucleic acid segment employing a unique sequence high complexity nucleic acid probe of greater than about 50,000 bases, wherein said targeted chromosomal material is a genetic rearrangement associated with at least one chromosome in humans, said method comprising employing said chromosomal material and a unique sequence high complexity nucleic acid probe of greater than about 50,000 bases in in situ hybrdization, wherein the chromosomal material is present in a morphologically identifiable cell nucleus; allowing said probe to bind to said targeted chromosomal material; and detecting said bound probe, wherein bound probe is indicative of the presence of target chromosomal material.
 2. A method of staining targeted interphase chromosomal material based upon a nucleic acid segment employing a unique sequence high complexity nucleic acid probe of greater than about 40 kb, wherein said targeted chromosomal material is a genetic rearrangement associated with at least one chromosome in humans, said method comprising contacting said chromosomal material with a unique sequence high complexity nucleic acid probe of greater than about 40 kb, wherein the chromosomal material is present in a morphologically identifiable cell nucleus; allowing said probe to bind to said targeted chromosomal material; and detecting said bound probe, wherein bound probe is indicative of the presence of target chromosomal material.
 3. A method of staining targeted interphase chromosomal material based upon a nucleic acid segment employing a unique sequence high complexity nucleic acid probe of greater than about 50,000 bases, wherein said targeted interphase chromosomal material is a genetic rearrangement associated with at least one chromosome in humans, said method comprising contacting said interphase chromosomal material with a unique sequence high complexity nucleic acid probe of greater than about 50,000 bases, wherein the chromosomal material is present in a morphologically identifiable cell nucleus; allowing said probe to bind to said targeted interphase chromosomal material; and detecting said bound probe, wherein bound probe is indicative of the presence of target interphase chromosomal material.
 4. The method of claim 2, wherein the genetic rearrangement is a translocation or an inversion.
 5. The method of claim 2, wherein the unique sequence high complexity nucleic acid probe is labeled.
 6. The method of claim 5, wherein said labeled unique sequence high complexity nucleic acid probe comprises fragments complementary to a single chromosome, fragments complementary to a subregion of a single chromosome, fragments complementary to a genome or fragments complementary to a subregion of a genome.
 7. The method of claim 2, wherein the interphase chromosomal material is interphase chromosomal DNA.
 8. The method of claim 3, wherein the genetic rearrangement is a translocation or an inversion.
 9. The method of claim 3, wherein the unique sequence high complexity nucleic acid probe is labeled.
 10. The method of claim 9, wherein said labeled unique sequence high complexity nucleic acid probe comprises fragments complementary to a single chromosome, fragments complementary to a subregion of a single chromosome, fragments complementary to a genome or fragments complementary to a subregion of a genome.
 11. The method of claim 3, wherein the interphase chromosomal material is interphase chromosomal DNA.
 12. The method of claim 2, wherein complexity of the unique sequence high complexity nucleic acid probe is greater than about 100,000 bases.
 13. The method of claim 3, wherein complexity of the unique sequence high complexity nucleic acid probe is greater than about 100,000 bases. 